Method for detecting aldehyde genotoxic impurities in atorvastatin calcium

Abstract

The invention provides a method for simultaneously detecting benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium, the method adopts a UPLC-TUV method for detection, genotoxic impurities benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium can be efficiently separated and detected, the method is strong in specificity, high in sensitivity and good in accuracy, and the method can be used for detecting the content of benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium. The method can be used for quality control of atorvastatin calcium.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for detecting aldehyde genotoxic impurities in atorvastatin calcium. Background Art

[0002] Atorvastatin is a new type of 3-hydroxy-2-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. It is a new type of highly effective lipid-lowering drug that can simultaneously reduce serum cholesterol and triglycerides. Compared with other similar statins, it has the advantages of wide application, good efficacy, and few side effects. Atorvastatin has attracted widespread attention from domestic pharmaceutical companies due to its wider indications, better tolerance and safety. The academic community has also conducted multi-faceted research on its production process, especially the synthesis of intermediates. Atorvastatin is generally used as its calcium salt. At present, the main synthesis route of its main chain structure M4 is to use isobutyrylacetanilide as a raw material to react with benzaldehyde for Knoevenagal condensation reaction, and then react with p-fluorobenzaldehyde for Setter reaction to obtain the product. In this synthesis process, due to the relationship between chemical equilibrium, it is inevitable that a small amount of benzaldehyde and p-fluorobenzaldehyde will remain. Aldehyde impurities have a high warning structure of genotoxic impurities, which is different from general impurities in drugs. They have significant safety risks and can induce DNA mutations at extremely trace levels. Therefore, the aldehyde genotoxic impurities introduced in the production process of atorvastatin calcium should be strictly controlled below the threshold of toxicological concern (TTC).

[0003] Patent CN 112213424A discloses a method for simultaneously determining coexisting impurities in an atorvastatin calcium intermediate, wherein the coexisting impurities of the parent ring M4 include M1, M2, M3, and M4 difluorinated impurities and M4 defluorinated impurities. Patent CN 114295768B discloses a method for determining 10 impurities in the mother nucleus M4 of atorvastatin, wherein the 10 impurities in the mother ring M4 are aniline, amino (4-methyl-3-oxo-N-phenylpentanamide), diphenylurea, fluorobenzene, acylate (4-fluorophenyl)-2-phenylethanone), bromide (2-bromo-1-(4-fluorophenyl)-2-phenylethanone), M3 (4-methyl-3-oxo-N-phenyl-2-(benzenyl)pentanamide), M4 defluorinated (2-(1,2-bis(4-fluorophenyl)-2-oxoethyl)-4-methyl-3-oxo-N-phenylpentylamine), M4 bisfluorinated (4-methyl-3-oxo-2-(2-oxo-1,2-diphenylethyl)-N-phenylpentanamide), dibromide (2 ,2-dibromo-1-(4-fluorophenyl)-2-phenylethan-1-one).

[0004] However, there are few reports on the detection methods of aldehyde genotoxic impurities benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium, and there is no method in the prior art that can simultaneously detect the above two potential genotoxic impurities. Therefore, it is necessary to provide a detection method for benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium to ensure product quality and ensure drug safety. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a detection method for simultaneously detecting benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium. The method can efficiently separate and detect the potential genotoxic impurities benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium, has strong specificity, high sensitivity and good accuracy, and can be used for the quality control of atorvastatin calcium.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: a method for detecting aldehyde genotoxic impurities in atorvastatin calcium, using UPLC-TUV method for detection, and using external standard quantitative method to calculate the contents of benzaldehyde and p-fluorobenzaldehyde respectively:

[0007] The chromatographic conditions of the UPLC-TUV method are as follows: using a reverse phase chromatographic column Waters Acquity UPLC BEH C18, 2.1 mm×50 mm, 1.7 μm, gradient elution with mobile phase A and mobile phase B, a flow rate of 0.29-31 ml / min, a column temperature of 39-41°C, detection wavelengths of 270 nm and 272 nm; and an injection volume of 8 μL.

[0008] The mobile phase A is 10 mM ammonium acetate aqueous solution, and the mobile phase B is acetonitrile.

[0009] The gradient elution procedure is:

[0010]

[0011] Furthermore, the reverse phase chromatography column is Waters Acquity UPLC BEH C18, 2.1 mm×50 mm, 1.7 μm.

[0012] Furthermore, the flow rate is 0.3 ml / min and the column temperature is 40°C.

[0013] Furthermore, the atorvastatin calcium treatment method is: weigh atorvastatin calcium, dissolve it completely with methanol, dilute it with an aqueous solution containing 15% acetonitrile to a solution with a concentration of 4 mg / ml, then ultrasonicate, centrifuge, and filter through a 0.2 μm filter membrane.

[0014] Beneficial effects of the present invention:

[0015] 1. The present invention establishes a method for rapidly determining the contents of genotoxic impurities benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium by UPLC-TUV. The method can simultaneously detect benzaldehyde and p-fluorobenzaldehyde, greatly improving the detection efficiency, and the reagents used are low in cost and have low requirements on chromatographic columns and instruments.

[0016] 2. The method established by the present invention can accurately determine the content of genotoxic impurities benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium, with simple operation, few influencing factors, good repeatability, short detection time, strong specificity, good specificity, high sensitivity and wide detection range of 15ng / ml-150ng / ml.

[0017] 3. The method established by the present invention is helpful for the market supervision of atorvastatin calcium, ensuring the quality and safety of drugs, and providing a technical basis for the establishment of a method for detecting genotoxic impurities in statins. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the sample detection spectrum of Example 1 of the present invention (from top to bottom, they are samples YHRN1802501, YHRN1802502, and YHRN1803501);

[0019] Figure 2 It is the detector selection comparison spectrum of Example 2 of the present invention;

[0020] Figure 3 This is a comparison chart of mobile phase elution gradient selection in Example 2 of the present invention;

[0021] Figure 4 This is the system suitability graph for the methodological verification of Example 3 of the present invention;

[0022] Figure 5 This is the specificity spectrum of the methodological verification of Example 3 of the present invention (top: blank-water; middle: blank-acetonitrile; bottom: blank-sample);

[0023] Figure 6 This is the sensitivity spectrum of the methodological verification of Example 3 of the present invention (left: detection limit; right: quantification limit);

[0024] Figure 7 This is the quantitative limit precision spectrum of the methodological verification of Example 3 of the present invention;

[0025] Figure 8 This is the linear test spectrum of the methodological verification of Example 3 of the present invention;

[0026] Fig. 9 This is a graph showing the results of a benzaldehyde linearity test for methodological verification in Example 3 of the present invention;

[0027] Fig.10This is a diagram showing the results of a linear test of p-fluorobenzaldehyde for the methodological verification of Example 3 of the present invention;

[0028] Fig.11 This is the accuracy (spiked 50%) spectrum of the methodological verification of Example 3 of the present invention;

[0029] Fig.12 This is the accuracy (spiked 100%) spectrum of the methodological verification of Example 3 of the present invention;

[0030] Fig.13 This is the accuracy (spiked 150%) spectrum of the methodological verification of Example 3 of the present invention;

[0031] Fig.14 This is the precision spectrum of the methodological verification of Example 3 of the present invention;

[0032] Fig.15 This is the intermediate precision spectrum of the methodological verification of Example 3 of the present invention;

[0033] Fig.16 This is the column temperature durability spectrum of the chromatographic column verified by the methodology of Example 3 of the present invention (top: 40°C; middle: 39°C; bottom: 41°C);

[0034] Fig.17 This is the flow rate durability spectrum of the methodological verification of Example 3 of the present invention (top: 0.30 ml / min; middle: 0.29 ml / min; bottom: 0.31 ml / min);

[0035] Fig.18 This is the stability (reference solution) spectrum of the methodological verification of Example 3 of the present invention;

[0036] Fig.19 This is the stability (test solution) spectrum of the methodological verification of Example 3 of the present invention. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with test examples and specific implementation methods. However, it should not be understood that the protection scope of the present invention is limited to the following embodiments, and all technologies implemented based on the present invention belong to the scope of the present invention.

[0038] Example 1 Detection of impurities benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium in the present invention

[0039] 1. Reagents

[0040] Reagents: acetonitrile (chromatographic grade, Fisher); ammonium acetate (chromatographic grade, JTBaker); Watsons water; methanol (chromatographic grade, Fisher); glacial acetic acid (chromatographic grade, Komiou).

[0041] Reference substances: benzaldehyde (chromatographically pure, McLean, purity ≥99.5%); p-fluorobenzaldehyde (chromatographically pure, McLean, purity 98%).

[0042] Test sample: Atorvastatin calcium (homemade, batch numbers: YHRN1802501, YHRN1802502, YHRN1803501).

[0043] 2. Determination of limits of benzaldehyde and p-fluorobenzaldehyde

[0044] Referring to the "Guidelines on Limits of Genotoxic Impurities" issued by EMA (European Medicines Evaluation Organization), the TTC (Threshold of Toxicological Concern) threshold is set at 1.5 μg / day. The maximum dose of atorvastatin calcium is 80 mg / day, based on the test sample of 4 mg / mL:

[0045]

[0046] The limits for benzaldehyde and p-fluorobenzaldehyde are both 75 ng / mL.

[0047] 3. Preparation of reference solution and test solution

[0048] (1) Stock solution a: Accurately weigh an appropriate amount of benzaldehyde reference substance and place it in a 10 mL volumetric flask. Add acetonitrile to dissolve and dilute to the mark to prepare a benzaldehyde stock solution of approximately 3 mg / mL.

[0049] (2) Stock solution b: Accurately weigh an appropriate amount of p-fluorobenzaldehyde reference substance and place it in a 10 mL volumetric flask. Add acetonitrile to dissolve and dilute to the mark to prepare a p-fluorobenzaldehyde stock solution of approximately 3 mg / mL.

[0050] (3) Mixed stock solution: Accurately measure 100 μL each of stock solutions a and b, place them in a 10 mL volumetric flask, dilute with methanol and make up to volume, shake well, and use this as the intermediate stock solution; accurately measure 250 μL of the intermediate stock solution in a 10 mL volumetric flask, dilute with methanol and make up to volume, shake well, and use this as the mixed stock solution.

[0051] (4) Reference solution: Accurately measure 1 mL of the mixed stock solution into a 10 mL volumetric flask, dilute with 15% acetonitrile aqueous solution and make up to volume. Shake well to prepare the reference solution.

[0052] (5) Preparation of test solution: Accurately weigh 40 mg of atorvastatin calcium and place it in a 10 mL volumetric flask. Add 1 mL of methanol and shake well to completely dissolve the sample. Dilute and make up to volume with 15% acetonitrile aqueous solution, shake well, sonicate for 2 min, centrifuge at 12,000 r / min for 5 min, and filter through a 0.2 μm microporous filter membrane to obtain the solution.

[0053] 4 Chromatographic conditions

[0054] Instrument: Waters Acquity UPLC ultra-high performance liquid chromatograph;

[0055] Detector: Waters TUV detector, detection wavelength: 270nm, 272nm;

[0056] Chromatographic conditions:

[0057] Chromatographic column: Waters Acquity UPLC BEH C18 column, 2.1 mm × 50 mm, 1.7 μm;

[0058] Column temperature: 40°C; Flow rate: 0.3 ml / min; Injection volume: 8 μL;

[0059] Mobile phase A: 10 mM ammonium acetate aqueous solution; Mobile phase B: acetonitrile;

[0060] Gradient elution:

[0061] Table 1 Gradient elution program

[0062]

[0063] 5. Detection

[0064] Accurately measure 8 µL of the reference solution and the test solution and inject them into the ultra-high performance liquid chromatograph respectively, record the chromatogram and the chromatographic peak area; calculate the contents of benzaldehyde and p-fluorobenzaldehyde in the sample by the external standard method.

[0065] 6 Experimental results

[0066] Three batches of atorvastatin calcium were tested. The results are shown in Table 2 and the spectra are shown in Figure 1 Benzaldehyde and p-fluorobenzaldehyde were not detected in the three batches of test samples.

[0067] Table 2 Test results of test products

[0068]

[0069] Example 2

[0070] 1. Optimize the detector

[0071] The same volume of reference solution was injected and the sensitivity of PDA detector and TUV detector were compared under the same chromatographic conditions. The chromatograms are shown in Figure 2 As shown in the figure, benzaldehyde and p-fluorobenzaldehyde of the same concentration had no response under the PDA detector. The PDA detector was replaced with a TUV detector, and the wavelength of the TUV detector was further optimized, which effectively improved the sensitivity. Finally, dual wavelength detection of 270 and 272 nm was selected.

[0072] 2. Optimize elution gradient

[0073] The reference solution was diluted to an appropriate concentration for injection. The separation of benzaldehyde and p-fluorobenzaldehyde under different elution gradients of the same mobile phase was compared under the same chromatographic conditions. The specific gradients are shown in Table 3 and the chromatograms are shown in Figure 3 .

[0074] Table 3 Different gradient elution programs

[0075]

[0076] like Figure 3 As shown, comparing the separation of benzaldehyde and p-fluorobenzaldehyde under the two elution gradients, gradient 1 is obviously better than gradient 2, so gradient 1 is selected for elution in this method.

[0077] 3. Optimize the mobile phase

[0078] The reference solution was injected separately, and the same elution gradient (gradient 1 in the above 2) was selected under the same chromatographic conditions to compare the separation of benzaldehyde and p-fluorobenzaldehyde under different mobile phases. Specifically, the peak shape, peak response and separation degree were compared when 10mM ammonium acetate aqueous solution, 0.1% formic acid aqueous solution were used as mobile phase A, and acetonitrile and methanol were used as mobile phase B. As a result, the separation degree was poor when 0.1% formic acid aqueous solution was used as mobile phase A; no peak was produced when methanol was used as mobile phase B; when 10mM ammonium acetate aqueous solution was used as mobile phase A and acetonitrile was used as mobile phase B, the peak response and peak shape were the best, and the separation degree was the best. Therefore, this method finally selected 10mM ammonium acetate aqueous solution as mobile phase A and acetonitrile as mobile phase B.

[0079] Example 3 Methodology Verification

[0080] 1 System suitability test

[0081] Take the reference solution and repeat 6 injections to calculate the peak area RSD. The results are shown in Table 4 and the spectrum is shown in Figure 4 .

[0082] Table 4 System suitability test results

[0083]

[0084] The results showed that the RSD of the benzaldehyde peak area after 6 repeated injections was 0.68%, and the RSD of the p-fluorobenzaldehyde peak area was 0.44%. Both RSDs were less than 10.0%, meeting the test requirements.

[0085] 2 Specificity test

[0086] Precisely measure 8 μL of blank solvent (acetonitrile, water) and test solution respectively and inject them into the ultra-high performance liquid chromatograph to record the chromatogram. Figure 5 .

[0087] The results showed that blank solvent and atorvastatin calcium did not interfere with the determination of benzaldehyde and p-fluorobenzaldehyde.

[0088] 3 Sensitivity

[0089] 3.1 Detection limit and quantification limit

[0090] The mixed reference solution of benzaldehyde and p-fluorobenzaldehyde was diluted and injected step by step, and the quantification limit and detection limit were set at a signal-to-noise ratio (S / N) of 10 and 3, respectively. The results are shown in Table 5. The chromatogram is shown in Figure 6 .

[0091] Table 5 Results of limit of quantitation and limit of detection

[0092]

[0093] 3.2 Quantitation limit precision test

[0094] Take the quantitative limit concentration solution, accurately measure 8 μL and continuously inject it into the ultra-high performance liquid chromatography instrument, record the chromatogram and peak area, repeat the injection 6 times, the results are shown in Table 6, and the spectrum is shown in Figure 7 .

[0095] Table 6 Quantitation limit precision results

[0096]

[0097] The results show that the sensitivity can meet the measurement requirements.

[0098] 4 Linearity and linear range

[0099] Accurately measure 5mL of mixed stock solution and place it in a 25mL volumetric flask, add 15% acetonitrile water to dilute and fix the volume as the standard stock solution; accurately measure 0.5mL, 1.3mL, 1.7mL, 2.5mL, and 4.2mL of the standard stock solution and place them in a 5mL volumetric flask, dilute to the scale with 15% acetonitrile water, shake well, and use them as the linear solutions of benzaldehyde and p-fluorobenzaldehyde L1-5 for standby use. The standard stock solution is used as the L6 linear solution. Take 8μL of each of the above linear solutions and inject them into the ultra-high performance liquid chromatograph, record the chromatogram and peak area. Perform linear regression analysis with the peak area as the ordinate and the concentration as the abscissa.

[0100] The results are shown in Tables 7-8, and the linear graphs are shown in Figures 9-10 . Spectrum see Figure 8 .

[0101] Table 7 Linearity and range of benzaldehyde determination

[0102]

[0103] Table 8 Linearity and range of p-fluorobenzaldehyde determination

[0104]

[0105] The results showed that the concentration of benzaldehyde was in the range of 15.08~150.84ng / ml, and it showed a good linear relationship with the peak area. The standard curve was Y=0.7128X+1.374, R 2 =0.9994, the percentage of the Y-axis intercept to the 100% response value is 0.35; the concentration of p-fluorobenzaldehyde is in the range of 14.91-149.11 ng / ml, and it shows a good linear relationship with the peak area. The standard curve is Y=0.7128X+1.374, R 2 =0.9994, the percentage of the Y-axis intercept to the 100% response value is 0.39. Each component has a good linear relationship within the corresponding concentration range.

[0106] 5 Accuracy test

[0107] Spiked test solution: Accurately weigh several portions of 40 mg atorvastatin calcium and place them in a 10 mL volumetric flask. Add 0.5 mL, 1.0 mL, and 1.5 mL of the mixed stock solution, respectively, dissolve and make up to volume with 15% acetonitrile aqueous solution, shake well, ultrasonicate for 2 min, centrifuge at 12000 r / min for 5 min, and filter through a 0.2 μm filter membrane to obtain 50%, 100%, and 150% spiked test solutions. Prepare three portions in the same way.

[0108] Take 8 μL of the reference solution and the spiked test solution and inject them into the ultra-high performance liquid chromatograph, record the chromatogram and the peak area of ​​each component, and calculate the recovery rate of each component by the external standard method. The results are shown in Tables 9-10. Figure 11~Figure 13 .

[0109] Table 9 Benzaldehyde recovery test results

[0110]

[0111] Table 10 p-Fluorobenzaldehyde recovery test results

[0112]

[0113] The results showed that at high, medium and low concentrations, the recovery rate of benzaldehyde content determination was between 104.9% and 114.4%, with an average recovery rate of 109.4% and an RSD of 3.04%; at high, medium and low concentrations, the recovery rate of p-fluorobenzaldehyde content determination was between 105.7% and 112.9%, with an average recovery rate of 108.4% and an RSD of 2.21%; the RSDs were all less than 10.0%, indicating that the method was accurate and reliable and could be used for the determination of benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium.

[0114] 6 Precision

[0115] 6.1 Repeatability

[0116] According to the accuracy test, six 100% spiked test solution were prepared, and 8 μL of the reference solution and spiked test solution were injected into the ultra-high performance liquid chromatograph, and the chromatogram and peak area of ​​each component were recorded. The recovery rate of each component was calculated by the external standard method. The results are shown in Table 11 and the spectra are shown in Fig.14 .

[0117] Table 11 Results of repeatability tests of benzaldehyde and p-fluorobenzaldehyde

[0118]

[0119] The results showed that the average benzaldehyde content was 110.6%, and the RSD was 2.86%; the average p-fluorobenzaldehyde content was 108.4%, and the RSD was 1.41%; the RSDs were all less than 10.0%, indicating that the method had good repeatability.

[0120] 6.2 Intermediate precision

[0121] Different personnel were used to conduct precision tests on different days. The results are shown in Table 12 and the spectra are shown in Fig.15 .

[0122] Table 12 Intermediate precision test results of benzaldehyde and p-fluorobenzaldehyde

[0123]

[0124] The results showed that the average content of benzaldehyde was 104.8%, and the RSD was 6.19%; the average content of p-fluorobenzaldehyde was 103.4%, and the RSD was 5.09%; the RSDs were all less than 10.0%, indicating that the intermediate precision of this method was good and could meet the requirements of content determination.

[0125] 7 Durability test

[0126] The influence of slight changes in the instrument liquid phase parameters on the determination of benzaldehyde and p-fluorobenzaldehyde content was investigated respectively. The same batch of samples were determined under various conditions. The results are shown in Tables 13-14 and the spectra are shown in Figures 16 and 17 .

[0127] Table 13 Benzaldehyde content durability test results

[0128]

[0129] Table 14 Results of durability test of p-fluorobenzaldehyde content

[0130]

[0131] The results show that after making appropriate changes to the liquid phase parameters, the determination results of benzaldehyde and p-fluorobenzaldehyde contents did not change significantly, and the RSDs were all less than 10.0%, indicating that the system conditions have good durability, and the determination data of benzaldehyde and p-fluorobenzaldehyde are stable and reliable, and can meet the requirements of the determination.

[0132] 8 Solution stability test

[0133] Under the accuracy item, prepare the reference solution and 100% spiked test solution, place at room temperature, take 8µL at 0h, 1h, 2h, 4h, 6h, and 8h and inject into the ultra-high performance liquid chromatograph, record the chromatogram and the peak area of ​​each component, calculate the RSD% of the peak area of ​​each component, the results are shown in Tables 15-16, and the spectra are shown in Figures 18 and 19 .

[0134] Table 15 Solution stability results of reference solution

[0135]

[0136] Table 16 Solution stability results of spiked test solution

[0137]

[0138] The results showed that when the reference solution was placed at room temperature for 8 hours, the RD of the benzaldehyde peak area was 1.03%, and the RD of the p-fluorobenzaldehyde peak area was 2.20%. Both RDs were less than 10.0%, indicating that the benzaldehyde and p-fluorobenzaldehyde reference solutions had good stability within 8 hours.

[0139] The spiked test solution was placed at room temperature for 8 hours. The RD of the benzaldehyde peak area was 1.57%, and the RD of the p-fluorobenzaldehyde peak area was 2.56%. Both RDs were less than 10.0%, indicating that the benzaldehyde and p-fluorobenzaldehyde in the spiked test solution were stable within 8 hours.

[0140] Based on the above experiments, the methodological validation shows that the established method has good specificity, sensitivity, precision, accuracy, durability and stability, and can accurately and reliably detect benzaldehyde and p-fluorobenzaldehyde in atorvastatin calcium, which helps to ensure drug safety and provide a technical basis for the establishment of a detection method for aldehyde genotoxic impurities in statins.

Claims

1. A method for detecting aldehyde genotoxic impurities in atorvastatin calcium, using UPLC-TUV method to detect and calculate the contents of benzaldehyde and p-fluorobenzaldehyde respectively: The chromatographic conditions of the UPLC-TUV method are as follows: using a reverse phase chromatographic column, gradient elution with mobile phase A and mobile phase B, a flow rate of 0.29-0.31 ml / min, a column temperature of 39-41°C, and detection wavelengths of 270 nm and 272 nm; The mobile phase A is 10 mM ammonium acetate aqueous solution, and the mobile phase B is acetonitrile; The gradient elution procedure is: 。 2. The detection method according to claim 1, characterized in that: The calculation of the contents of benzaldehyde and p-fluorobenzaldehyde is to calculate the contents of benzaldehyde and p-fluorobenzaldehyde respectively by peak area external standard quantitative method.

3. The method according to claim 3, characterized in that: The reverse phase chromatography column is Waters Acquity UPLCBEH C18, 2.1 mm×50 mm, 1.7 μm.

4. The method according to claim 4, characterized in that: The flow rate was 0.3 ml / min and the column temperature was 40°C.

5. The method according to claim 1, characterized in that The atorvastatin calcium treatment method is as follows: weigh atorvastatin calcium, dissolve it completely with methanol, dilute it with an aqueous solution containing 15% acetonitrile to a solution with a concentration of 4 mg / ml, then ultrasonicate, centrifuge, and filter through a 0.2 μm filter membrane.

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