Reversed phase chromatography method for measuring atorvastatin calcium enantiomer

Abstract

The invention discloses a reversed-phase chromatography method for determining atorvastatin calcium enantiomers, which can directly and effectively separate atorvastatin calcium and enantiomers, has good specificity and precision, high sensitivity and good durability, and is simpler and more convenient to operate, easier to balance, lower in cost and more environment-friendly compared with the existing normal-phase chromatography. The method can be directly used for detecting atorvastatin calcium and the preparation thereof, and is more convenient to operate, so that better guarantee is provided for quality control of atorvastatin calcium and the preparation thereof.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for analyzing and determining the enantiomers of atorvastatin calcium and its preparations by reverse phase liquid chromatography. Background Art

[0002] Atorvastatin was first synthesized in 1985 by Bruce Roth of Parke-Davis Warner-Lambert Company (now Pfizer) and was first approved for marketing in the UK in November 1996 as tablets (trade name: Lipitor). The currently approved specifications are 10, 20, 40, and 80 mg for hypercholesterolemia and prevention of cardiovascular disease. It was subsequently approved for marketing in many other European countries through a non-centralized approval process. In December 1996 and March 2000, atorvastatin calcium tablets were approved for marketing in the United States and Japan, respectively. As of August 2022, Lipitor has been approved for marketing in more than 100 countries around the world. Atorvastatin is currently a product of Viatris, a company formed by the merger of Pfizer's business unit Pfizer Upjohn and Mylan.

[0003] Atorvastatin calcium oral suspension (trade name: Atorvaliq, specification: 20mg / 5ml) was developed by the pharmaceutical company CMPDEVELOPMENT LLC and was first approved by the FDA on February 1, 2023 for the treatment of hypercholesterolemia and coronary heart disease. In addition, atorvastatin calcium is also available in dispersible tablets, capsules and other dosage forms.

[0004] The chemical name of atorvastatin calcium is (3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-isopropylpyrrol-1-yl]-3,5-dihydroxyheptanoate calcium (2:1) trihydrate, and the structural formula is as follows:

[0005]

[0006] Atorvastatin calcium has two chiral centers. The chemical name of its enantiomer is (3S,5S)-7-[2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoate calcium (2:1). The structural formula is as follows:

[0007]

[0008] As potential degradation impurities, enantiomers of atorvastatin calcium may affect the quality of the final product atorvastatin calcium and its preparations. By consulting Chinese and foreign literature and patents, it was found that relevant literature has reported the use of high performance liquid chromatography to determine the optical enantiomers of atorvastatin calcium and its intermediates, and basically all of them use normal phase chromatography systems.

[0009] CN104931599A relates to a method for determining related substances of atorvastatin calcium, comprising the following steps: step 1, preparation of a test solution: taking an appropriate amount of atorvastatin calcium, adding a solvent to dissolve and quantitatively diluting to prepare a solution containing about 1 mg per 1 ml, as the test solution; step 2, preparation of a mixed reference solution: weighing an appropriate amount of reference substances of impurity A, impurity B, impurity C, impurity D, impurity E and an atorvastatin calcium reference substance, adding the above solvent to dissolve and quantitatively diluting to prepare a solution containing about 3 μg of impurity A, 2 μg of impurity B, impurity C, impurity D, impurity E and 10 μg of atorvastatin calcium per 1 ml, as the mixed reference solution; step 3, high performance liquid chromatography HPLC analysis: taking 20 μl of the test solution and the mixed reference solution, respectively injecting them into a liquid chromatograph, recording the chromatogram until the end of the gradient elution program, and calculating the content of each component according to the peak area of ​​the chromatogram. The inventors have found that the reverse phase chromatography system can only separate diastereomers but cannot separate enantiomers.

[0010] CN113702514A discloses a method for determining impurity I related to atorvastatin calcium, which belongs to the field of drug impurity detection. The technical scheme thereof includes the following steps: (a) preparing a test solution: weighing atorvastatin calcium, dissolving it with a solvent and quantitatively diluting it to prepare a solution containing 0.8-1.2 mg per 1 mL, as the test solution; (b) preparing a mixed reference solution: weighing an impurity A reference, an impurity B reference, an impurity C reference, an impurity D reference, an impurity E reference, an impurity I reference, and an atorvastatin calcium reference, dissolving them with the above-mentioned solvent and quantitatively diluting them to prepare a solution containing 54-66 μg of impurity B, 5-15 μg each of impurities A, impurity C, impurity D, impurity E, and impurity I, and 45-55 μg of atorvastatin calcium per 1 mL, as the mixed reference solution; and (c) analyzing the test solution and the mixed reference solution by high performance liquid chromatography. The present inventors have found that the reverse phase chromatography system can only separate diastereomers but not enantiomers.

[0011] Therefore, it is necessary to establish a highly sensitive, simple, and universal detection method for the determination of atorvastatin calcium enantiomers. Summary of the invention

[0012] The present invention solves the problems existing in the prior art and provides a reverse phase chromatography method for determining the enantiomers of atorvastatin calcium. The method is simple to operate, has good specificity and precision, and has high sensitivity, good method durability, and high general applicability. The method can be used for the quality control of enantiomers in atorvastatin calcium and related preparations, and meets existing needs.

[0013] The invention provides a reverse phase chromatography method for determining enantiomers of atorvastatin calcium, comprising the following steps: respectively sampling a system suitability solution and a test solution, detecting according to high performance liquid chromatography conditions, and recording a chromatogram; the chromatographic conditions are: a chromatographic column: a chromatographic column with cellulose-tris (3-chloro-4-methylphenylcarbamate) silica gel as a filler; and a mobile phase: a buffer solution and an organic solvent.

[0014] The chromatographic conditions also include:

[0015] Detector: UV detector;

[0016] Flow rate: 0.4~0.6 ml / min, preferably 0.5 ml / min;

[0017] Column temperature: 25~35℃, preferably 30℃;

[0018] Detection wavelength: 244nm;

[0019] Injection volume: 20 μl.

[0020] The chromatographic column of the present invention is selected from Daicel Chiralcel OZ-RH, 4.6mm×150mm, 5μm, ChiralMZ(2)-RH 4.6mm×150mm, 5μm, Lux Cellulose-2 4.6mm×150mm, 5μm or a chromatographic column with equivalent performance, preferably Daicel Chiralcel OZ-RH, 4.6mm×150mm, 5μm.

[0021] The buffer solution in the mobile phase of the present invention is selected from one or more of acetate buffer, phosphate buffer and formate buffer, the acetate buffer is selected from glacial acetic acid and ammonium acetate, the phosphate buffer is selected from phosphoric acid and potassium dihydrogen phosphate, and the formate buffer is selected from formic acid and ammonium formate; preferably, the buffer solution is acetate buffer, the acetate buffer is preferably glacial acetic acid, and the concentration of glacial acetic acid is 0.5%~1.5% (ml / ml), preferably 1% (ml / ml). The organic solvent is selected from one or more of acetonitrile and methanol, preferably, the organic solvent is acetonitrile. The volume ratio of the buffer solution and the organic solvent is 60~70:30~40, preferably 65:35.

[0022] The system suitability solution is prepared by the following process: weighing appropriate amounts of atorvastatin calcium and atorvastatin calcium enantiomer reference substances, adding a solvent to dissolve and dilute to prepare the system suitability solution.

[0023] Furthermore, the system suitability solution is prepared by the following process: taking appropriate amounts of atorvastatin calcium reference substance and enantiomer reference substance, dissolving and diluting them with a solvent to prepare a mixed solution containing approximately 0.1 mg of atorvastatin and 0.75 μg of enantiomer per 1 ml.

[0024] The test solution is prepared by the following process: taking an appropriate amount of atorvastatin calcium or its preparation, adding a solvent to dissolve and dilute to prepare the test solution. The preparation includes oral suspension, tablets, dispersible tablets, capsules, etc.

[0025] Furthermore, the test solution is prepared by the following process: take an appropriate amount of atorvastatin calcium oral suspension (equivalent to 10 mg of atorvastatin), accurately weigh it, put it in a 100 ml volumetric bottle, add an appropriate amount of solvent, shake it to dissolve it, dilute it to the scale with the solvent, shake it well, filter it, and take the filtrate. The solvent is acetonitrile and water mixed in equal volumes, shake it well to obtain the solution.

[0026] The present invention further provides a method for determining the enantiomers of atorvastatin calcium by reverse phase chromatography and its use in the quality control of atorvastatin calcium and its preparations.

[0027] The beneficial technical effects of the present invention are:

[0028] (1) The analytical method of the present invention can well separate atorvatin calcium from its enantiomers. The reverse phase chromatography system used has the advantages of stable chromatographic solvent performance, strong separation ability, high sensitivity, simple operation process, easier balance, more environmentally friendly and economical relative to normal phase solvents, and stronger universality.

[0029] (2) The analytical method of the present invention can be directly used for the detection of atorvastatin calcium and its liquid preparations. The operation is more convenient and efficient. It avoids the situation where the liquid preparations use normal phase solvents that are immiscible and need to undergo a complicated extraction pre-treatment process. It simplifies the processing steps and can more conveniently achieve quality control of the product production process, improve the quality of the final product, and ensure the safety and effectiveness of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the system suitability test spectrum in the separation test of Example 2.

[0031] Figure 2 This is the sensitivity test spectrum in the separation test of Example 2.

[0032] Figure 3 This is the test spectrum of the test sample in the separation test of Example 2.

[0033] Figure 4 This is the system suitability test spectrum of Example 3.

[0034] Figure 5 This is the system suitability test spectrum of Example 4.

[0035] Figure 6 This is the system suitability test spectrum of Example 5.

[0036] Figure 7 This is the system suitability test spectrum of Example 6.

[0037] Figure 8 This is the system suitability test spectrum of Example 7.

[0038] Fig. 9 This is the system suitability test spectrum of Example 8.

[0039] Fig.10 This is the system suitability test spectrum of Example 9.

[0040] Fig.11 This is the system suitability test spectrum of Example 10.

[0041] Fig.12 This is the precision test spectrum of Example 11. DETAILED DESCRIPTION

[0042] Specific embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0043] Instrument and chromatographic conditions:

[0044] Instrument: Waters ARC liquid chromatograph, UV detector;

[0045] Flow rate: 0.5ml / min;

[0046] Column temperature: 30°C;

[0047] Detection wavelength: 244nm;

[0048] Injection volume: 20 µl.

[0049] Solution preparation:

[0050] System suitability solution: Take appropriate amounts of atorvastatin calcium reference substance and enantiomer reference substance, dissolve in solvent and dilute to prepare a mixed solution containing approximately 0.1 mg of atorvastatin and 0.75 μg of enantiomer per 1 ml.

[0051] Test solution: Take an appropriate amount of atorvastatin calcium oral suspension (approximately equivalent to 10 mg of atorvastatin), accurately weigh, place in a 100 ml volumetric flask, add an appropriate amount of solvent, shake to dissolve, dilute to the scale with solvent, shake well, filter, and take the filtrate.

[0052] Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with a solvent to prepare a solution containing approximately 0.3 μg of atorvastatin per 1 ml.

[0053] Sensitivity solution: Accurately measure an appropriate amount of the control solution and quantitatively dilute it with a solvent to prepare a solution containing approximately 0.15 μg of atorvastatin per 1 ml.

[0054] Solvent: Mix equal volumes of acetonitrile and water and shake well;

[0055] Example 1 Screening of Chromatographic Conditions

[0056] On the basis of the above chromatographic conditions, the chromatographic conditions were screened according to Table 1. The separation results of atorvastatin calcium and enantiomers showed that the chromatographic conditions of test condition 9 had good peak shapes for both peaks, and the separation between the main peak and the enantiomer peak achieved good results.

[0057] Table 1 Chromatographic conditions screening

[0058] ;

[0059] Example 2 Separation Test

[0060] The experiment was continued with the chromatographic conditions of Test Condition 9 in Example 1.

[0061] Accurately measure 20 μl of system suitability solution and sensitivity solution, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Tables 2, 3 and Figure 1 , Figure 2 The separation between the main peak and the enantiomer chromatographic peak should meet the requirements, and the signal-to-noise ratio of the main component peak height should not be less than 10. Accurately measure 20μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 4 and Figure 3 If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the enantiomer, the enantiomer peak area shall not be greater than the main peak area of ​​the reference solution (0.3%) calculated by the main component self-reference method.

[0062] Table 2 System suitability solution test results

[0063]

[0064] Table 3 Sensitivity solution test results

[0065]

[0066] Table 4 Test results of test solution

[0067]

[0068] Results: In the system suitability solution chromatogram, atorvastatin and enantiomers eluted sequentially, and the separation between the two peaks was greater than 1.5. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height was greater than 10. In the test solution chromatogram, the detection amount of enantiomers met the requirements.

[0069] Example 3 System suitability test

[0070] The instrument and chromatographic conditions were the same as above, and the mobile phase was 1% (ml / ml) glacial acetic acid-acetonitrile (70:30).

[0071] Preparation of mobile phase: Take 1% (ml / ml) glacial acetic acid and acetonitrile, mix them in a ratio of (70:30), shake well, and you have the mobile phase.

[0072] Take the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. Figure 4 .

[0073] Results: In the system suitability solution chromatogram, the main peak and the enantiomer peaks were well separated.

[0074] Example 4 System suitability test

[0075] The instrument and chromatographic conditions were the same as above, and the mobile phase was 1% (ml / ml) glacial acetic acid-acetonitrile (60:40).

[0076] Preparation of mobile phase: Take 1% (ml / ml) glacial acetic acid and acetonitrile, mix them in a ratio of (60:40), shake well, and you have the mobile phase.

[0077] Take the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. Figure 5 .

[0078] Results: In the system suitability solution chromatogram, the main peak and the enantiomer peaks were well separated.

[0079] Example 5 System suitability test

[0080] The instrument and chromatographic conditions were the same as above, and the mobile phase was 0.5% (ml / ml) glacial acetic acid-acetonitrile (65:35).

[0081] Preparation of mobile phase: Take 0.5% (ml / ml) glacial acetic acid and acetonitrile, mix them in a ratio of (65:35), shake well, and you have the mobile phase.

[0082] Take the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. Figure 6 .

[0083] Results: In the system suitability solution chromatogram, the main peak and the enantiomer peaks were well separated.

[0084] Example 6 System suitability test

[0085] The instrument and chromatographic conditions were the same as above, and the mobile phase was 1.5% (ml / ml) glacial acetic acid-acetonitrile (65:35).

[0086] Preparation of mobile phase: Take 1.5% (ml / ml) glacial acetic acid and acetonitrile, mix them in a ratio of (65:35), shake well, and you have the mobile phase.

[0087] Take the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. Figure 7 .

[0088] Results: In the system suitability solution chromatogram, the main peak and the enantiomer peaks were well separated.

[0089] Example 7 System suitability test

[0090] The instrument and chromatographic conditions were the same as above, and the column temperature was 25°C.

[0091] Preparation of mobile phase: Take 1% (ml / ml) glacial acetic acid and acetonitrile, mix them in a ratio of (65:35), shake well, and you have the mobile phase.

[0092] Take the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. Figure 8 .

[0093] Results: In the system suitability solution chromatogram, the main peak and the enantiomer peaks were well separated.

[0094] Example 8 System suitability test

[0095] The instrument and chromatographic conditions were the same as above, and the column temperature was 35°C.

[0096] Preparation of mobile phase: Take 1% (ml / ml) glacial acetic acid and acetonitrile, mix them in a ratio of (65:35), shake well, and you have the mobile phase.

[0097] Take the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. Fig. 9 .

[0098] Results: In the system suitability solution chromatogram, the main peak and the enantiomer peaks were well separated.

[0099] Example 9 System suitability test

[0100] The instrument and chromatographic conditions were the same as above, with a flow rate of 0.4 ml / min.

[0101] Preparation of mobile phase: Take 1% (ml / ml) glacial acetic acid and acetonitrile, mix them in a ratio of (65:35), shake well, and you have the mobile phase.

[0102] Take the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. Fig.10 .

[0103] Results: In the system suitability solution chromatogram, the main peak and the enantiomer peaks were well separated.

[0104] Example 10 System suitability test

[0105] The instrument and chromatographic conditions were the same as above, with a flow rate of 0.6 ml / min.

[0106] Preparation of mobile phase: Take 1% (ml / ml) glacial acetic acid and acetonitrile, mix them in a ratio of (65:35), shake well, and you have the mobile phase.

[0107] Take the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. Fig.11 .

[0108] Results: In the system suitability solution chromatogram, the main peak and the enantiomer peaks were well separated.

[0109] Based on the above test results, under the chromatographic conditions of Examples 2 to 10, the main peak and the enantiomers appeared in sequence, and the separation degree between the two peaks was not less than 1.5, which met the requirements.

[0110] Example 11 Precision Test

[0111] Prepare 6 test sample solutions of the same concentration, accurately measure 20 μl of each, inject into the liquid chromatograph, record the chromatogram, and calculate the detection amount of each isomer and the relative standard deviation of the detection amount. The results are shown in Table 5 and Fig.12 .

[0112] Table 5 Results of precision test of enantiomers of atorvastatin calcium oral suspension

[0113] .

Claims

1. A reverse phase chromatography method for determining enantiomers of atorvastatin calcium, characterized in that: The method comprises the following steps: taking a system suitability solution and a test solution for injection respectively, testing according to high performance liquid chromatography conditions, and recording a chromatogram; the chromatographic conditions are: a chromatographic column: a chromatographic column with cellulose-tris (3-chloro-4-methylphenylcarbamate) silica gel as a filler; Mobile phase: acetate buffer and acetonitrile, wherein the test sample is atorvastatin calcium oral suspension.

2. The reverse phase chromatography method for determining the enantiomers of atorvastatin calcium according to claim 1, characterized in that: The chromatographic conditions also include: Detector: UV detector; Flow rate: 0.4~0.6 ml / min; Column temperature: 25~35℃; Detection wavelength: 244nm; Injection volume: 20 μl.

3. The reverse phase chromatography method for determining the enantiomers of atorvastatin calcium according to claim 1, characterized in that: The chromatographic column is selected from Daicel Chiralcel OZ-RH, 4.6mm×150mm, 5μm, Chiral MZ(2)-RH 4.6mm×150mm, 5μm, and Lux ​​Cellulose-2 4.6mm×150mm, 5μm.

4. The reverse phase chromatography method for determining the enantiomers of atorvastatin calcium according to claim 1, characterized in that: The concentration of the acetate buffer is 0.5%-1.5% (ml / ml).

5. The reverse phase chromatography method for determining the enantiomers of atorvastatin calcium according to claim 1, characterized in that: The volume ratio of the acetate buffer to acetonitrile is 60-70:30-40.

6. The reverse phase chromatography method for determining enantiomers of atorvastatin calcium according to claim 1, characterized in that: The system suitability solution is prepared by the following process: taking appropriate amounts of atorvastatin calcium and atorvastatin calcium enantiomer reference substances, dissolving and diluting them with a solvent to prepare a system suitability solution; the test solution is prepared by the following process: taking appropriate amounts of atorvastatin calcium or its preparation, dissolving and diluting them with a solvent to prepare a test solution.

7. The reverse phase chromatography method for determining enantiomers of atorvastatin calcium according to claim 6, characterized in that: The solvent is a mixture of acetonitrile and water in equal volumes.

8. Use of the reverse phase chromatography method for determining the enantiomers of atorvastatin calcium according to claim 1 in the quality control of atorvastatin calcium and its preparations.

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