A method for separating pitavastatin calcium starting material and its impurities.

Abstract

This invention relates to the field of analytical chemistry, specifically to a method for separating pitavastatin calcium starting material and its impurities. The separation method of this invention uses paraformaldehyde as a derivatizing reagent to replace the triphenylphosphine group, amplifying the positional isomerism differences of fluorophenyl groups, thereby achieving effective separation of pitavastatin calcium starting material and its impurities. It has the advantages of high separation degree, good repeatability, strong specificity, high sensitivity, good accuracy and strong stability.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for separating pitavastatin calcium starting material and its impurities. Background Technology

[0002] Pitavastatin calcium, chemically named (+)-bis-{(3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolinyl]-3,5-dihydroxy-6-enheptanoic acid} monocalcium salt, is used clinically as an inhibitor of hydroxymethylglutaryl-CoA (HMG-CoA) reductase to treat hyperlipidemia.

[0003] Common pitavastatin calcium preparations typically use [[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolinyl]methyl]triphenylphosphine bromide as a starting material, for example: CN103508948A. The preparation of [[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolinyl]methyl]triphenylphosphine bromide usually uses anthranilic acid as a starting material, which is obtained through chlorination, Friedel-Crafts alkylation, condensation, reduction, substitution, and salt formation with triphenylphosphine. In the Friedel-Crafts alkylation reaction, in addition to the target compound at the para position, defluorination, meta-, and ortho-isomers are inevitably generated. These defluorination, meta-, and ortho-isomers are introduced into the pitavastatin calcium production process through the Wetting reaction, directly participating in subsequent synthesis processes. The polarity of the product is close to that of the pitavastatin calcium intermediate, making it difficult to remove in subsequent processes, ultimately transforming into defluorination, ortho-, and meta-isomer impurities in the finished pitavastatin calcium product.

[0004] The chemical name of the starting material for pitavastatin calcium is: [[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolinyl]methyl]triphenylphosphine bromide, CAS: 154057-58-6, and its structural formula is as follows: C 37 H 30 BrFNP, molecular weight: 618.53.

[0005] Impurity A is [(2-cyclopropyl-4-phenyl-3-quinolinyl)methyl]triphenylphosphine bromide, a defluorination impurity of this starting material; impurity B is [[2-cyclopropyl-4-(3-fluorophenyl)-3-quinolinyl]methyl]triphenylphosphine bromide; and impurity C is [[2-cyclopropyl-4-(2-fluorophenyl)-3-quinolinyl]methyl]triphenylphosphine bromide, its fluorine positional isomer. The structural formulas of the three are as follows: The presence of the above three impurities can affect the detection of the content of the starting material. Although there are reports on methods for separating the above compounds from their impurities, such as CN115166104B and CN115290791B, these separation methods have problems such as poor separation degree and poor accuracy. Therefore, there is an urgent need to provide a method for separating pitavastatin calcium starting material and its impurities with high separation degree and good accuracy. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for separating pitavastatin calcium starting material and its impurities, so as to solve the problems of poor separation degree and poor accuracy existing in the prior art.

[0007] This invention is achieved through the following technical solution:

[0008] This invention provides a method for separating pitavastatin calcium starting material and its impurities, characterized in that the separation method employs derivatization followed by liquid chromatography, and includes the following steps;

[0009] 1) Preparation of the derivatized solution of the test sample, the derivatized solution of the reference standard, and the 100% spiked derivatized solution of the test sample:

[0010] A) Preparation of the derivatization solution of the test sample: Take a certain weight of pitavastatin calcium starting material test sample, place it in a volumetric flask, carry out the derivatization reaction using derivatization method 1, and then prepare a test sample derivatization solution with a concentration of about 0.2 mg / mL using dimethyl sulfoxide solution and / or diluent.

[0011] B) Preparation of reference standard derivatization solution: Accurately measure a certain amount of mixed impurity stock solution, place it in a volumetric flask, carry out derivatization reaction using derivatization method 1, and then prepare a reference standard derivatization solution with a concentration of approximately 0.2 μg / mL using dimethyl sulfoxide solution and / or diluent.

[0012] C) Preparation of 100% spiked test sample derivatization solution: Take a certain weight of pitavastatin calcium starting material test sample and a certain volume of mixed impurity stock solution and carry out derivatization reaction using derivatization method 1. Then, prepare a 100% spiked test sample derivatization solution containing 0.2 mg pitavastatin calcium starting material test sample, 0.2 μg pitavastatin calcium impurity A derivative, 0.2 μg calcium impurity C derivative and 0.2 μg calcium impurity B derivative per 1 mL using dimethyl sulfoxide solution and / or diluent.

[0013] 2). Take 20 μL of the solution prepared in step 1) and inject it into the liquid chromatography system for qualitative or quantitative analysis at a wavelength of 245 nm.

[0014] The reaction formula for the derivative method 1 is:

[0015] The R is selected from: H or F;

[0016] The specific steps of derivative method 1 are as follows: accurately weigh a certain weight of pitavastatin calcium starting material, impurities A, B and / or C, place them in a volumetric flask, add a certain amount of dimethyl sulfoxide to dissolve, add potassium carbonate and paraformaldehyde, place a magnetic stirrer to stir and heat at 80°C for 1 hour, take it out and let it cool to room temperature, dilute with dimethyl sulfoxide to the mark, and shake well.

[0017] The preparation method of the mixed impurity stock solution is as follows: accurately measure a certain amount of impurity A stock solution, impurity C stock solution and impurity B stock solution respectively, place them in a volumetric flask, add dimethyl sulfoxide to dilute to the mark, shake well, and the solution is obtained.

[0018] The structural formula of the pitavastatin calcium starting material is as follows:

[0019] The impurities include: impurity A, impurity B and impurity C;

[0020] The structure of impurity A is as follows:

[0021] The structure of impurity B is as follows:

[0022] The structure of the impurity C is as follows:

[0023] Furthermore, as a preferred embodiment of the present invention, the analytical conditions for the liquid chromatography are as follows:

[0024] Column: Agilent InfinityLab Poroshell 120PFP, 4.6mm × 250mm, 4μm;

[0025] Mobile phase A: Measure 1 mL of phosphoric acid, add 1000 mL of water, mix well, and adjust the pH to 3.0 with triethylamine to obtain the mobile phase (or prepare it according to the ratio).

[0026] Mobile phase B: Acetonitrile

[0027] Flow rate: 0.8-1.2 mL / min;

[0028] Column temperature: 38-42℃;

[0029] Detection wavelength: 245nm;

[0030] Injection volume: 20 μL;

[0031] Elution ratio: The volume ratio of mobile phase A to mobile phase B is 55:45.

[0032] Furthermore, as a preferred embodiment of the present invention, the analytical conditions for the liquid chromatography are as follows:

[0033] Column: Agilent InfinityLab Poroshell 120PFP, 4.6mm × 250mm, 4μm;

[0034] Mobile phase A: Measure 1 mL of phosphoric acid, add 1000 mL of water, mix well, and adjust the pH to 3.0 with triethylamine to obtain the mobile phase (or prepare it according to the ratio).

[0035] Mobile phase B: Acetonitrile

[0036] Flow rate: 1.0 mL / min;

[0037] Column temperature: 40℃;

[0038] Detection wavelength: 245nm;

[0039] Injection volume: 20 μL;

[0040] Elution ratio: The volume ratio of mobile phase A to mobile phase B is 55:45.

[0041] Furthermore, as a preferred embodiment of the present invention, the derivatization reaction time in step 1) is 50 min-70 min; preferably, the derivatization reaction time in step 1) is 1 h.

[0042] Furthermore, as a preferred embodiment of the present invention, in step A), the mass ratio of pitavastatin calcium starting material: potassium carbonate: paraformaldehyde is 4:3:3;

[0043] Furthermore, as a preferred embodiment of the present invention, in step B), the volume-to-mass ratio of the mixed impurity stock solution: potassium carbonate: paraformaldehyde is 1 mL: 15 mg: 15 mg.

[0044] Furthermore, as a preferred embodiment of the present invention, in step C), the volume-to-mass ratio of the mixed impurity stock solution: pitavastatin calcium starting material: potassium carbonate: paraformaldehyde is 1 mL: 20 mg: 15 mg: 15 mg.

[0045] Furthermore, as a preferred embodiment of the present invention, the volume ratio of impurity A stock solution: impurity C stock solution: impurity B stock solution in the mixed impurity stock solution is 1:1:1;

[0046] Furthermore, as a preferred embodiment of the present invention: the preparation of the test sample derivatization solution, the reference standard derivatization solution, and the 100% spiked test sample derivatization solution in step 1) is as follows:

[0047] A) Derivatized solution of the test sample

[0048] Weigh approximately 20 mg of pitavastatin calcium starting material into a 5 mL volumetric flask, add 2 mL of dimethyl sulfoxide to dissolve it, then add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it into a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain a derivatized solution of the test sample with a concentration of approximately 0.2 mg / mL.

[0049] B) Reference standard derivatized solution

[0050] Accurately measure 1 mL of the mixed impurity stock solution and place it in a 5 mL volumetric flask. Add 1 mL of dimethyl sulfoxide, 15 mg of potassium carbonate, and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain a reference standard derivatized solution with a concentration of about 0.2 μg / mL.

[0051] C) 100% spiked test sample derivatized solution

[0052] Weigh approximately 20 mg of pitavastatin calcium starting material into a 5 mL volumetric flask. Accurately add 1 mL of mixed impurity stock solution and 1 mL of dimethyl sulfoxide to dissolve the sample. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove the sample and allow it to cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for approximately 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well.

[0053] Furthermore, as a preferred technical solution of the present invention, the preparation method of the impurity A stock solution, impurity C stock solution and impurity B stock solution is as follows: take about 5 mg of each of impurity A, impurity C and impurity B reference standards, weigh them accurately, place them in 10 mL volumetric flasks, add dimethyl sulfoxide to dissolve and dilute to the mark, shake well, and use them as impurity A stock solution, impurity C stock solution and impurity B stock solution respectively.

[0054] Furthermore, as a preferred technical solution of the present invention, the preparation method of the mixed impurity stock solution is as follows: accurately measure 1 mL of impurity A stock solution, impurity C stock solution and impurity B stock solution respectively, place them in a 25 mL volumetric flask, dilute with dimethyl sulfoxide to the mark, and shake well to obtain the solution;

[0055] Furthermore, as a preferred technical solution of the present invention, the preparation method of the mixed impurity stock solution is as follows: take about 5 mg each of impurity A reference standard, impurity B reference standard, and impurity C reference standard, accurately weigh them, place them in a 10 mL volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the mark, and shake well; accurately measure 1 mL, place it in a 25 mL volumetric flask, add dimethyl sulfoxide to dilute to the mark, and shake well to obtain the solution.

[0056] Furthermore, as a preferred embodiment of the present invention, the present invention also provides a method for separating pitavastatin calcium starting material and its impurities, characterized in that the separation method employs post-derivative liquid chromatography, comprising the following steps:

[0057] 1) Derivatization and preparation of solutions:

[0058] A) Blank derivatized solution

[0059] Take 2 mL of dimethyl sulfoxide and place it in a 5 mL volumetric flask. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove and cool to room temperature. Dilute with dimethyl sulfoxide to the mark, shake well, and let stand for about 5 minutes. Accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add diluent to the mark and shake well.

[0060] B) Derivatized solution of the test sample

[0061] Accurately weigh approximately 20 mg of pitavastatin calcium starting material and place it in a 5 mL volumetric flask. Add 2 mL of dimethyl sulfoxide to dissolve it, then add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add diluent to the mark and shake well to obtain a derivatized solution of the test sample with a concentration of approximately 0.2 mg / mL.

[0062] C) Reference standard derivatized solution

[0063] Accurately measure 1 mL of the mixed impurity stock solution and place it in a 5 mL volumetric flask. Add 1 mL of dimethyl sulfoxide, 15 mg of potassium carbonate, and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add diluent to the mark and shake well to obtain a reference standard derivatized solution with a concentration of about 0.2 μg / mL.

[0064] D) 100% spiked test sample derivatized solution

[0065] Accurately weigh approximately 20 mg of the test sample and place it in a 5 mL volumetric flask. Accurately add 1 mL of mixed impurity stock solution and 1 mL of dimethyl sulfoxide to dissolve the sample. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove the sample and allow it to cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for approximately 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add diluent to the mark and shake well to obtain the final product.

[0066] 2) Take 20 μL of the solution prepared in step 1) and inject it into the liquid chromatography system for qualitative or quantitative analysis at a wavelength of 245 nm.

[0067] Furthermore, as a preferred embodiment of the present invention, the diluent is a 50% (volume ratio) acetonitrile-water solution.

[0068] Furthermore, as a preferred embodiment of the present invention, the present invention also provides a derivative prepared by the derivation method 1 according to the present invention, characterized in that the derivative is selected from the following structures:

[0069]

[0070] Furthermore, as a preferred embodiment of the present invention, the present invention also provides the application of the derivative prepared by the derivation method 1 according to the present invention as a reference in the separation and detection of pitavastatin calcium starting material and its impurities.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] 1) This invention employs a pre-column derivatization method, using paraformaldehyde as a derivatizing reagent to replace the triphenylphosphine group, thereby amplifying the positional isomerism differences of fluorophenyl groups. This achieves effective separation of pitavastatin calcium starting material and its impurities, and has the advantages of high separation degree, good repeatability, strong specificity, high sensitivity, good accuracy and strong stability.

[0073] 2) The derivative method of the present invention is simple and easy to operate. Attached image description:

[0074] 1) Figure 1 Specificity overlay of pitavastatin calcium starting material and impurity derivatives;

[0075] 2) Figure 2 The graph shows the linearity of the derivative of impurity A.

[0076] 3) Figure 3 Linear graph of impurity C derivative;

[0077] 4) Figure 4 The graph shows the linearity of the derivative of impurity B.

[0078] 5) Figure 5 This is a liquid chromatography overlay of Comparative Example 1;

[0079] 6) Figure 6 This is a superimposed liquid chromatography image of Comparative Example 2;

[0080] 7) Figure 7 The mass spectrum of the derivative of the test sample;

[0081] 8) Figure 8 This is the mass spectrum of the derivative of impurity A;

[0082] 9) Figure 9 This is the mass spectrum of the derivative of impurity B;

[0083] 10) Figure 10 This is the mass spectrum of the C derivative of impurity. Detailed Implementation

[0084] The present invention will be further described in detail below with reference to the embodiments, but the content of the invention is not limited to the embodiments.

[0085] Test sample information

[0086]

[0087] Reference Standard Information

[0088] Impurity A Shenzhen Hengfeng Wanda 20211201 95.80％ Impurity C Shenzhen Hengfeng Wanda 20190501 98.33％ Impurity B Shenzhen Hengfeng Wanda 20190501 94.65％

[0089] Instruments and chromatographic columns

[0090]

[0091] Example 1

[0092] 1. Solution preparation

[0093] 1) Mobile phase: 0.1% aqueous phosphoric acid solution (adjusted to pH 3.0 with triethylamine) - acetonitrile (55:45)

[0094] Measure 1 mL of phosphoric acid and add it to 1000 mL of water. Mix well and adjust the pH to 3.0 with triethylamine. Measure 550 mL of the above solution and add 450 mL of acetonitrile. Mix well to obtain the final product. (Alternatively, prepare according to the specified proportions.)

[0095] 2) Diluent / Blank Solution: Acetonitrile-Water (50:50, volume ratio)

[0096] 3) Test solution

[0097] Weigh approximately 10 mg of pitavastatin calcium starting material accurately, dissolve it in diluent and bring the volume to the mark to prepare a test solution with a concentration of approximately 0.1 mg / mL.

[0098] 4) Localization solutions of impurities A, B, and C

[0099] Take approximately 5 mg each of impurity A, impurity B, and impurity C reference standards, accurately weigh them, dissolve them in diluent, and dilute to the mark to prepare a positioning solution of impurity A, impurity B, and impurity C with a concentration of approximately 0.1 μg / mL.

[0100] 5) Mixed impurity stock solution

[0101] Weigh approximately 5 mg each of impurity A reference standard, impurity B reference standard, and impurity C reference standard accurately, dissolve them in diluent and bring the volume to the mark to prepare a mixed impurity stock solution with a concentration of approximately 10 μg / mL.

[0102] 6) 100% spiked test solution

[0103] Weigh approximately 10 mg of pitavastatin calcium starting material accurately, add 1 mL of mixed impurity stock solution, dissolve with diluent and bring to volume to prepare a 100% spiked test solution.

[0104] 7) Blank derivatization solution

[0105] Take 2 mL of dimethyl sulfoxide and place it in a 5 mL volumetric flask. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove and cool to room temperature. Dilute with dimethyl sulfoxide to the mark, shake well, and let stand for about 5 minutes. Accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well.

[0106] 8) Derivatized solution of test sample

[0107] Weigh approximately 20 mg of pitavastatin calcium starting material into a 5 mL volumetric flask, add 2 mL of dimethyl sulfoxide to dissolve it, then add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it into a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain a derivatized solution of the test sample with a concentration of approximately 0.2 mg / mL.

[0108] 9) Derivatized localization solutions of impurities A, B, and C

[0109] Accurately weigh approximately 5 mg each of impurity A, impurity B, and impurity C reference standards, and place them separately in 10 mL volumetric flasks. Dissolve and dilute each flask with dimethyl sulfoxide to the mark, and mix well. These are the stock solutions of impurity A, impurity B, and impurity C. Accurately measure 1 mL of each stock solution and place them separately in 25 mL volumetric flasks. Dilute each flask with dimethyl sulfoxide to the mark, and mix well. Accurately measure 1 mL of each stock solution and place them separately in 5 mL volumetric flasks. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde to 1 mL of the solution, stir with a magnetic stirrer and heat at 80 °C for 1 hour. After removing from the heat and cooling to room temperature, dilute with dimethyl sulfoxide to the mark and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain the derivatized localization solutions of impurity A, impurity B and impurity C with a concentration of about 0.2 μg / mL.

[0110] 10) Reference standard derivatized solution

[0111] Accurately measure 1 mL of the mixed impurity stock solution and place it in a 5 mL volumetric flask. Add 1 mL of dimethyl sulfoxide, 15 mg of potassium carbonate, and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain a reference standard derivatized solution with a concentration of about 0.2 μg / mL.

[0112] 11) 50% spiked test sample derivatization solution

[0113] Weigh approximately 20 mg of the test sample accurately and place it in a 5 ml volumetric flask. Accurately add 0.5 ml of mixed impurity stock solution and 1.5 ml of dimethyl sulfoxide to dissolve the sample. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove the sample and allow it to cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for approximately 5 minutes, accurately measure 1 ml of the supernatant and place it in a 20 ml volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well.

[0114] 12) 100% spiked test sample derivatization solution

[0115] Accurately weigh approximately 20 mg of the test sample and place it in a 5 mL volumetric flask. Accurately add 1 mL of mixed impurity stock solution and 1 mL of dimethyl sulfoxide to dissolve the sample. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove the sample and allow it to cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for approximately 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain the final product.

[0116] 13) 150% spiked test sample derivatization solution

[0117] Weigh approximately 20 mg of the test sample accurately and place it in a 5 ml volumetric flask. Accurately add 1.5 ml of mixed impurity stock solution and 0.5 ml of dimethyl sulfoxide to dissolve the sample. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove the sample and allow it to cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for approximately 5 minutes, accurately measure 1 ml of the supernatant and place it in a 20 ml volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well.

[0118] 2. Experimental procedure: Inject 20 μl each of the blank derivatization solution, the derivatization and localization solutions of impurity A, impurity B and impurity C, the reference derivatization solution and the 100% spiked test sample derivatization solution, and record the chromatograms.

[0119] 3. Chromatographic conditions

[0120] Table 1

[0121]

[0122]

[0123] Example 2 Specificity

[0124] One part each of the blank derivatization solution, reference derivatization solution, derivatization and localization solution for impurity A, impurity B, and impurity C prepared in Example 1, one part of the test sample derivatization solution, and one part of the 100% spiked test sample derivatization solution were taken. Except for the reference derivatization solution, which was injected 5 times, each of the other solutions was injected once. One injection of the reference derivatization solution was injected back into the end of the sequence. Specific chromatographic conditions are shown in Table 1. Isocratic elution was performed, and the chromatograms were recorded. The results are shown in Tables 2 and 3. Figure 1 .

[0125] Table 2. Results of the suitability of the impurity specificity testing system

[0126]

[0127] Table 3 Results of impurity specificity tests

[0128]

[0129] [Note] The data for the 100% spiked test solution are referenced from the data for the first 100% spiked test solution under the repeatability section.

[0130] The experimental results show that the blank derivatization solution does not interfere with the detection. In the reference derivatization solution, the minimum resolution between the peaks of each target impurity derivative is 5.6; the maximum RSD of the peak area of ​​each target impurity derivative in the five injections and all reference derivatization solutions is 1.8%. In the 100% spiked test solution, the retention times of each target impurity derivative peak are consistent with those in the positioning solutions; the minimum resolution between each target impurity derivative peak and adjacent peaks, and between the main component derivative peak and adjacent peaks, is 3.3; the recovery rates of the detection results for each target impurity are all between 80% and 120%. This method has good specificity.

[0131] Example 3 Linear

[0132] Using the limit of quantitation (LOQ) concentration as the lowest point of the linear range, a mixed impurity stock solution was taken, diluted with an appropriate amount of dimethyl sulfoxide (DMSO), and 15 mg of potassium carbonate and 15 mg of paraformaldehyde were added. The mixture was stirred with a magnetic stirrer and heated at 80°C for 1 hour. After being removed and cooled to room temperature, the solution was diluted to the mark with DMSO and shaken well. After standing for about 5 minutes, 1 mL of the supernatant was accurately measured and placed in a 20 mL volumetric flask. The solution was diluted to the mark with diluent and shaken well to prepare a series of solutions equivalent to 30%–200% of the limit concentration. These solutions were used as linear solutions and injected into the liquid chromatograph for isocratic elution under the same chromatographic conditions as in Table 1. The chromatograms were recorded. The relationship between different concentrations of each target impurity and peak area was investigated together with the limit of quantitation solution. Linear regression was performed using the least squares method, with a correlation coefficient r not less than 0.998, the ratio of the Y-axis intercept to the 100% response value not exceeding 25%, and the response factor (A / C) RSD ≤ 10%. The sum of squared residuals was reported, and a residual plot was attached, with residual points distributed on both sides of the X-axis. The results are shown in Tables 4 to 6.

[0133] Table 4. Linearity test results of impurity A derivative.

[0134]

[0135]

[0136] Table 5. Linearity test results of impurity C derivative.

[0137]

[0138] Table 6. Linearity test results of impurity B derivative.

[0139]

[0140] The experimental results show that: in the concentration range of 0.0202 μg / mL to 0.4038 μg / mL, the impurity A derivative is equivalent to 0.0101% to 0.2019% of the test sample concentration; in the concentration range of 0.0201 μg / mL to 0.4021 μg / mL, the impurity C derivative is equivalent to 0.0101% to 0.2011% of the test sample concentration; and in the concentration range of 0.0197 μg / mL to 0.3947 μg / mL, the impurity B derivative is equivalent to 0.0099% to 0.1973% of the test sample concentration. The linearity of this method is good.

[0141] Example 4 Limit of Quantitation / Limit of Detection

[0142] Limit of Quantification

[0143] The reference standard derivatized solution was injected into a liquid chromatography system with isocratic elution under the same chromatographic conditions as in Table 1, ensuring the main peak height was 10 times the noise peak height. This concentration was used as the quantitation concentration, and the limit of quantitation (LOQ) was calculated. The signal-to-noise ratio of the LOQ solution should be no less than 10. After six repeated injections, the RSD of the peak area should be ≤10%. The LOQ concentration should not exceed 30% of the limit (0.06 μg / mL). The results are shown in Table 7.

[0144] Table 7 Results of Limit of Quantitation Tests for Impurity Derivatives

[0145]

[0146]

[0147] Experimental results show that this method has good sensitivity and can effectively quantify target impurities.

[0148] Detection limit

[0149] The reference standard derivatized solution was injected into the liquid chromatography system and isocratic eluted under the same chromatographic conditions as in Table 1, ensuring that the peak height of the main peak was three times the height of the noise peak. This concentration was then used as the minimum detection concentration, and the minimum detectable amount was calculated. The results are shown in Table 8.

[0150] Table 8 Results of the test for the detection limit of impurity derivatives

[0151]

[0152] Experimental results show that this method has good sensitivity and can effectively detect target impurities.

[0153] Example 5 Precision

[0154] Repeatability

[0155] Analyst 1 prepared one blank derivatization solution, one reference derivatization solution, and six 100% spiked test sample derivatization solutions. Except for the reference derivatization solution, which was injected five times, each of the other solutions was injected once. One injection of the reference solution was then injected back into the end of the sequence. Isocratic elution was performed under the same chromatographic conditions as in Table 1, and the chromatograms were recorded. The RSD or range of the detection amounts of each target impurity in the six 100% spiked test sample derivatization solutions should meet the requirements in the table below:

[0156] C% < 0.03% (or reporting limit) No comparison 0.03% (or reporting limit) ≤ C% < 0.10% Range ≤ 0.02% 0.10％≤C％＜0.2％ RSD ≤ 15% 0.2％≤C％＜0.5％ RSD≤10% 0.5％≤C％＜2％ RSD≤5% 2％≤C％ RSD≤2%

[0157] The results are shown in Tables 9 and 10.

[0158] Table 9 Results of the repeatability test system for impurity derivatives

[0159]

[0160] Table 10 Results of repeatability tests on impurity derivatives

[0161]

[0162]

[0163] The experimental results show that the blank derivatization solution does not interfere with the detection. In the reference derivatization solution, the minimum resolution between the peaks of each target impurity derivative is 5.6; the maximum RSD of the peak area of ​​each target impurity derivative in the five injections and all reference derivatization solutions is 1.8%. In the six 100% spiked test sample derivatization solutions, the RSD of the detection amount of each target impurity meets the validation requirements, and the method has good repeatability.

[0164] Example 6 Intermediate Precision

[0165] Analyst 2 performed repeatability tests on different dates using different liquid chromatographs. The detection limits and RSDs of each target impurity in six 100% spiked derivatized solutions of the test sample were calculated and compared with the repeatability results. The RSDs or ranges of the detection limits of each target impurity in the six 100% spiked derivatized solutions of the test sample by Analyst 2, and the RSDs or ranges of the detection limits of each target impurity in the twelve 100% spiked derivatized solutions of the test sample by the two analysts, should meet the requirements of the following table:

[0166]

[0167] The results are shown in Tables 11 and 12.

[0168] Table 11 Results of the applicability of the intermediate precision test system for impurity derivatives

[0169]

[0170] Table 12 Results of intermediate precision tests on impurity derivatives

[0171]

[0172] The experimental results show that the blank derivatization solution does not interfere with the detection. In the reference derivatization solution, the minimum resolution between the peaks of each target impurity derivative is 6.4; the maximum RSD of the peak area of ​​each target impurity derivative in the five needles and all reference derivatization solutions is 0.6%. In the six 100% spiked test sample derivatization solutions of Analyst 2, the RSD of the detection amount of each target impurity met the validation requirements, indicating that the intermediate precision of this method is good.

[0173] The RSDs of the detection amounts of each target impurity in 12 100% spiked test sample derivatization solutions by two analysts all met the validation requirements, indicating that the method has good precision.

[0174] Example 7 Accuracy

[0175] Take one part blank derivatization solution, one part reference derivatization solution, one part test sample derivatization solution, three parts 50% spiked test sample derivatization solutions, three parts 100% spiked test sample derivatization solutions, and three parts 150% spiked test sample derivatization solutions. Inject the reference derivatization solution five times, and inject each of the other solutions once. Inject one part of the reference derivatization solution back into the sequence. Perform chromatographic injections according to the chromatographic conditions in Table 1 and record the chromatograms. The results are shown in Tables 13 to 16.

[0176] Table 13 Results of the applicability of the repeatability test system for impurity derivatives

[0177]

[0178] Table 14. Accuracy test results of impurity A derivative.

[0179]

[0180] Table 15 Accuracy Test Results of Impurity C Derivative

[0181]

[0182]

[0183] Table 16. Accuracy test results of impurity B derivative.

[0184]

[0185] The experimental results show that the blank derivatization solution does not interfere with the detection. In the reference derivatization solution, the minimum resolution between the peaks of each target impurity derivative is 6.4; the maximum RSD of the peak area of ​​each target impurity derivative in the five injections and all reference derivatization solutions is 0.6%. In the nine spiked test solutions, the recoveries of each target impurity derivative are all within the range of 90%–110%; the RSDs of the recoveries of each target impurity derivative meet the validation requirements, indicating that the method has good accuracy.

[0186] Example 8 Solution Stability

[0187] Prepare one blank derivatization solution, one reference derivatization solution, and one 100% spiked test sample derivatization solution. Place the reference derivatization solution and the 100% spiked test sample derivatization solution at room temperature (10℃~30℃) under light-protected conditions, and inject them at different time points according to the chromatographic conditions in Table 1. Record the chromatograms. Relative to the stability test solution after 0 hours, the absolute value of the change rate of the peak area of ​​each target impurity derivative in the reference derivatization solution at each stability time point should be ≤10.0%; the absolute value of the change rate / absolute value of the detection amount of each target impurity in the 100% spiked test sample derivatization solution at each stability time point should meet the requirements in the table below:

[0188] Measured value < 0.03% (or reporting limit) The absolute value of the change is ≤0.01%. 0.03% (or reporting limit) ≤ measured value < 0.10% The absolute value of the change is ≤0.02%. 0.10% ≤ Measured value < 0.2% The absolute value of the rate of change is ≤30%. 0.2% ≤ Measured value < 0.5% The absolute value of the rate of change is ≤20%. 0.5% ≤ measured value < 2.0% The absolute value of the rate of change is ≤10%. 2.0% ≤ Measured value absolute value of the rate of change ≤ 5%

[0189] The results are shown in Tables 17 and 18.

[0190] Table 17 Results of stability tests on reference standard derivatized solutions

[0191]

[0192]

[0193] [Note] "——" indicates that it is not applicable. The 0-hour stability data are based on the results of the first reference solution under the specificity section.

[0194] Table 18 Results of Stability Tests on Derivatized Solutions of 100% Spiced Test Samples

[0195]

[0196] [Note] "——" indicates that it is not applicable.

[0197] The test results show that the reference solution is stable for at least 28 hours when placed at room temperature (10℃~30℃) without light protection. The 100% spiked derivatized solution of the test sample is stable for at least 24 hours when placed at room temperature (10℃~30℃) without light protection.

[0198] Example 9 Durability

[0199] The effects of varying flow rate, column temperature, and derivatization time on the detection results were investigated. The varying parameters are shown in the table below:

[0200] Flow rate 1.0 mL / min 0.8mL / min and 1.2mL / min Column temperature 40℃ 38℃ and 42℃ Derivative Time 1 hour (60 minutes) 50 minutes and 70 minutes

[0201] Each time, one condition was changed while keeping the others constant. One blank derivatization solution, one reference derivatization solution, and one 100% spiked test sample derivatization solution were prepared. Except for the reference derivatization solution, which was injected 5 times, each of the other solutions was injected once. One injection was then injected back into the reference derivatization solution at the end of the sequence. The chromatograms were recorded. The results are shown in Tables 19 and 20.

[0202] Table 19 Results of the robustness test system for impurity derivatives

[0203]

[0204] [Note] The robustness of the standard chromatographic conditions (flow rate 1.0 mL / min, column temperature 40℃, derivatization time 60 min) refers to the results of the system usability test under the intermediate precision section.

[0205] Table 20 Results of Impurity Derivative Impurity Durability Tests

[0206]

[0207] [Note] The robustness of the standard chromatographic conditions (flow rate 1.0 mL / min, column temperature 40 °C, derivatization time 60 min) is based on the results of the first 100% spiked test solution under the Intermediate Precision section.

[0208] The experimental results show that, under slight changes in chromatographic and derivatization conditions (flow rate 0.8 mL / min to 1.2 mL / min, column temperature 38℃ to 42℃, derivatization time 50 min to 70 min), the RSDs of the detection amounts of each target impurity in the 100% spiked test solution all meet the validation requirements, indicating that the method has good robustness.

[0209] Example 10 Sample Detection

[0210] Prepare one blank derivatization solution, one reference derivatization solution, and four batches of samples (batch numbers D2202091, N8.2-C231101, N8.2-C231102, and N8.2-C231103). Prepare one test sample derivatization solution for each batch of samples. Perform liquid chromatography analysis according to the chromatographic conditions in Table 1 and record the chromatograms. The results are shown in Tables 21 and 22.

[0211] Table 21 Results of the Applicability Test of the Impurity Derivative Sample Detection System

[0212]

[0213] Table 22 Detection results of impurity derivative samples

[0214]

[0215] Conclusion: The blank derivatization solution did not interfere with the detection. In the reference derivatization solution, the minimum resolution between the peaks of each target impurity derivative was 5.6; the maximum RSD of the peak area of ​​each target impurity derivative in the five needles and all reference derivatization solutions was 1.8%. The impurities detected in all four batches of samples met the acceptable standards.

[0216] Example 11: Structural identification of the test sample derivative and impurity derivative:

[0217] 11.1 Instrument / Materials Information:

[0218] Column numbers: QCL400, QCL295

[0219] High Performance Liquid Chromatograph (HPLC) Model: Waters H-Class; Registration Number: QC-OB03-006; Calibration Expiration Date: July 17, 2025

[0220] Liquid chromatography-mass spectrometry (LC-MS) instrument model: Agilent 6470, serial number: QC-LCMS-001, calibration expiration date: April 17, 2025.

[0221] 11.1.1 Chromatographic conditions:

[0222] Column: Waters XBridge BEH C18, 100mm × 3mm, 2.5μm

[0223] Mobile phase A: 0.1% formic acid aqueous solution

[0224] Mobile phase B: Acetonitrile

[0225] Mobile phase: Mobile phase A: Mobile phase B: = 40:60

[0226] Flow rate: 0.5 ml / min

[0227] Column temperature: 40℃

[0228] Injection volume: 20 μL

[0229] Running time: 15min

[0230] 11.1.2 Mass spectrometry conditions:

[0231] Ion source: APCI, positive mode

[0232] Scan mode: MS2 Scan

[0233] Data collection period: 3–15 min

[0234] 11.2 Solution preparation:

[0235] Blank solution / diluent: 50% acetonitrile-aqueous solution

[0236] 11.2.1 Test Derivative Solution: Accurately weigh approximately 20 mg of pitavastatin calcium starting material and place it in a 5 ml volumetric flask. Add 2 ml of dimethyl sulfoxide to dissolve it, then add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. Let stand for approximately 5 minutes. Measure 0.5 ml of the above solution and place it in a colorimetric tube. Add 3 ml of ethyl acetate and 10 ml of ultrapure water. Shake to separate the layers. Take 0.5 ml of the upper layer and place it in a 10 ml volumetric flask. Vacuum the flask to evaporate the solvent. Add 2 ml of diluent to dissolve the remaining residue.

[0237] Diluent for the test derivative: Measure 0.5 ml of the above-mentioned test derivative solution and place it in a 10 ml volumetric flask. Add diluent to the mark and mix well. Perform LC-MS analysis on the prepared diluent for the test derivative. The LC-MS results are as follows: Figure 7 As shown.

[0238] 11.2.2 Impurity A Derivative Solution: Take approximately 5 mg of impurity A reference standard and place it in a 10 ml volumetric flask. Dissolve and dilute to the mark with dimethyl sulfoxide (DMSO), and shake well to prepare the stock solution. Accurately measure 1 ml of the stock solution and place it in a 5 ml volumetric flask. Add 1 ml of DMSO, 15 mg of potassium carbonate, and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove and cool to room temperature. Dilute to the mark with DMSO and shake well. Let stand for approximately 5 minutes. Measure 0.5 ml of the above solution and place it in a colorimetric tube. Add 3 ml of ethyl acetate and 10 ml of ultrapure water. Shake to separate the layers. Take 0.5 ml of the upper layer and place it in a 10 ml volumetric flask. Vacuum the solution to evaporate the solvent. Add 2 ml of diluent to dissolve the remaining residue.

[0239] Diluent for Impurity A Derivative: Measure 0.5 ml of the above Impurity A derivative solution and place it in a 10 ml volumetric flask. Add diluent to the mark and mix well. Perform LC-MS analysis on the prepared Impurity A derivative diluent. The LC-MS results are as follows: Figure 8 As shown.

[0240] 11.2.3 Impurity B Derivative Solution: Take approximately 5 mg of impurity B reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, and shake well to prepare the stock solution. Accurately measure 1 ml of the stock solution, place it in a 5 ml volumetric flask, add 1 ml of dimethyl sulfoxide, 15 mg of potassium carbonate, and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove and cool to room temperature, dilute to the mark with dimethyl sulfoxide, and shake well. Let stand for approximately 5 minutes. Measure 0.5 ml of the above solution, place it in a colorimetric tube, add 3 ml of ethyl acetate and 10 ml of ultrapure water, shake to separate the layers, take 0.5 ml of the upper layer solution, place it in a 10 ml volumetric flask, vacuum to evaporate the solvent, add 2 ml of diluent to dissolve the remaining residue, and obtain the final solution.

[0241] Diluent for Impurity B Derivative: Measure 0.5 ml of the above Impurity B derivative solution and place it in a 10 ml volumetric flask. Add diluent to the mark and mix well. Perform LC-MS analysis on the prepared Impurity B derivative diluent. The LC-MS results are as follows: Figure 9 As shown.

[0242] 11.2.4 Impurity C Derivative Solution: Take approximately 5 mg of impurity C reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with dimethyl sulfoxide, and shake well to prepare the stock solution. Accurately measure 1 ml of the stock solution, place it in a 5 ml volumetric flask, add 1 ml of dimethyl sulfoxide, 15 mg of potassium carbonate, and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80°C for 1 hour. Remove and cool to room temperature, dilute to the mark with dimethyl sulfoxide, and shake well. Let stand for approximately 5 minutes. Measure 0.5 ml of the above solution, place it in a colorimetric tube, add 3 ml of ethyl acetate and 10 ml of ultrapure water, shake to separate the layers, take 0.5 ml of the upper layer solution, place it in a 10 ml volumetric flask, vacuum to evaporate the solvent, add 2 ml of diluent to dissolve the remaining residue, and obtain the final solution.

[0243] Impurity C derivative diluent: Measure 0.5 ml of the above impurity C derivative solution and place it in a 10 ml volumetric flask. Add diluent to the mark and mix well. Perform LC-MS analysis on the prepared impurity C derivative diluent. The LC-MS results are as follows: Figure 10 As shown.

[0244] Comparative Example 1

[0245] Sample preparation: Using the diluent as a blank solution, take 1 part of the test solution, 1 part of the impurity reference solution, and 1 part of the 100% spiked test solution for testing.

[0246] Experimental procedure: Inject 20 μL each of the blank solution, test solution, reference solution, and 100% spiked test solution, and analyze according to the chromatographic conditions in Table 1. Record the chromatograms. Results are shown below. Figure 5.

[0247] according to Figure 5 The results show that all impurities can be separated, but impurity B cannot be completely separated from the main peak. This indicates that without derivatization, even under the chromatographic conditions of this invention, it is difficult to separate the pitavastatin calcium starting material sample from the impurities.

[0248] Comparative Example 2

[0249] Derivative Method 2:

[0250] The R is selected from: H or F;

[0251] The specific steps of derivative method 2 are as follows: accurately weigh a certain amount of pitavastatin calcium starting material, impurities A, B and / or C, place them in a volumetric flask, add a certain amount of dimethyl sulfoxide to dissolve them, add potassium carbonate and acetone, stir with a magnetic stirrer and heat at 80°C for 1 hour, remove and cool to room temperature, dilute with dimethyl sulfoxide to the mark, and shake well.

[0252] Sample preparation:

[0253] 1) Blank derivatized solution 2

[0254] Take 2 mL of dimethyl sulfoxide and place it in a 5 mL volumetric flask. Add 15 mg of potassium carbonate and 60 μL of acetone. Stir with a magnetic stirrer and heat at 80 °C for 5 hours. Remove and cool to room temperature. Dilute to the mark with dimethyl sulfoxide, shake well, and let stand for about 5 minutes. Accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well.

[0255] 2) Derivatized solution of the test sample 2

[0256] Accurately weigh approximately 20 mg of pitavastatin calcium starting material and place it in a 5 mL volumetric flask. Add 2 mL of dimethyl sulfoxide to dissolve it, then add 15 mg of potassium carbonate and 60 μL of acetone. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain a derivatized solution of the test sample with a concentration of approximately 0.2 mg / mL.

[0257] 3) Derivatized localization solutions of impurities A, B, and C 2

[0258] Accurately weigh approximately 5 mg each of impurity A, impurity B, and impurity C reference standards, and place them separately in 10 mL volumetric flasks. Dissolve and dilute each flask with dimethyl sulfoxide to the mark, and mix well. These are the stock solutions of impurity A, impurity B, and impurity C. Accurately measure 1 mL of each stock solution and place them separately in 25 mL volumetric flasks. Dilute each flask with dimethyl sulfoxide to the mark, and mix well. Accurately measure 1 mL of each stock solution and place them separately in 5 mL volumetric flasks. Add 15 mg of potassium carbonate and 60 μL of acetone to 1 mL of the solution, stir with a magnetic stirrer and heat at 80 °C for 5 hours. After removing from the heat and cooling to room temperature, dilute with dimethyl sulfoxide to the mark and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain derivatized positioning solutions 2 for impurities A, B and C with a concentration of about 0.2 μg / mL.

[0259] 4) Reference standard derivatized solution 2

[0260] Accurately measure 1 mL of the mixed impurity stock solution and place it in a 5 mL volumetric flask. Add 1 mL of dimethyl sulfoxide, 15 mg of potassium carbonate, and 60 μL of acetone. Stir with a magnetic stirrer and heat at 80 °C for 5 hours. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for about 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent and dilute to the mark. Shake well to obtain a reference standard derivatized solution 2 with a concentration of about 0.2 μg / mL.

[0261] 5) 100% spiked test sample derivatization solution 2

[0262] Accurately weigh approximately 20 mg of the test sample and place it in a 5 mL volumetric flask. Accurately add 1 mL of mixed impurity stock solution and 1 mL of dimethyl sulfoxide to dissolve the sample. Add 15 mg of potassium carbonate and 60 μL of acetone. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove the sample and allow it to cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for approximately 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and / or diluent to the mark and shake well to obtain the final product.

[0263] 6) Experimental Procedure: Inject 20 μL each of the following solutions: blank derivatization solution 2, test sample derivatization solution 2, derivatization and localization solutions 2 for impurities A, B, and C, reference derivatization solution 2, and 100% spiked test sample derivatization solution 2. Analyze according to the chromatographic conditions in Table 1 and record the chromatograms. Results are shown in [Table 1]. Figure 6 .

[0264] according to Figure 6 The results show that, using the above-mentioned derivation method 2, impurities and main components can be completely separated, but a large number of by-products are generated, which interfere with the detection.

[0265] It should be understood that the above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims of the present invention.

Claims

1. A quantitative analysis method for pitavastatin calcium starting materials, characterized in that, The quantitative analysis method employs post-derivative liquid chromatography and includes the following steps: 1) Preparation of the derivatized solution of the test sample, the derivatized solution of the reference standard, and the 100% spiked derivatized solution of the test sample: A) Preparation of the derivatization solution of the test sample: Take a certain weight of pitavastatin calcium starting material test sample, place it in a volumetric flask, carry out the derivatization reaction using derivatization method 1, and then prepare a test sample derivatization solution with a concentration of 0.2 mg / mL using dimethyl sulfoxide solution. B) Preparation of reference standard derivatization solution: Accurately measure a certain amount of mixed impurity stock solution, place it in a volumetric flask, carry out derivatization reaction using derivatization method 1, and then prepare a reference standard derivatization solution with a concentration of 0.2 μg / mL using dimethyl sulfoxide solution; C) Preparation of 100% spiked test sample derivatization solution: Take a certain weight of pitavastatin calcium starting material test sample and a certain volume of mixed impurity stock solution and carry out derivatization reaction using derivatization method 1. Then, use dimethyl sulfoxide solution to prepare a 100% spiked test sample derivatization solution containing 0.2 mg pitavastatin calcium starting material derivative, 0.2 μg impurity A derivative, 0.2 μg impurity C derivative and 0.2 μg impurity B derivative per 1 mL. 2) Take 20 μL of the solution prepared in step 1) and inject it into the liquid chromatography system for quantitative analysis at a wavelength of 245 nm. The reaction formula for the derivation method 1 is as follows: The R is selected from: H or F; The specific steps of derivative method 1 are as follows: accurately weigh a certain weight of pitavastatin calcium starting material, impurities A, B and / or C, place them in a volumetric flask, add a certain amount of dimethyl sulfoxide to dissolve, add potassium carbonate and paraformaldehyde, place the flask in a magnetic stirrer and heat at 80°C for 1 hour, remove and cool to room temperature, dilute with dimethyl sulfoxide to the mark, and shake well. The preparation method of the mixed impurity stock solution is as follows: accurately measure a certain amount of impurity A stock solution, impurity C stock solution and impurity B stock solution respectively, place them in a volumetric flask, add dimethyl sulfoxide to dilute to the mark, shake well, and the solution is obtained. The derivatization reaction in step 1) takes 50-70 minutes; In step A), the mass ratio of pitavastatin calcium starting material: potassium carbonate: paraformaldehyde is 4:3:

3. In step B), the volume-to-mass ratio of the mixed impurity stock solution: potassium carbonate: paraformaldehyde is 1 mL: 15 mg: 15 mg. In step C), the volume-to-mass ratio of the mixed impurity stock solution, pitavastatin calcium starting material, potassium carbonate, and paraformaldehyde is 1 mL: 20 mg: 15 mg: 15 mg. The volume ratio of impurity A stock solution to impurity C stock solution to impurity B stock solution in the mixed impurity stock solution is 1:1:

1. The pitavastatin calcium starting material was sourced from Jiangxi Eifim Technology Co., Ltd., batch number D2202091; The analytical conditions for the liquid chromatography are as follows: Column: Agilent InfinityLab Poroshell 120 PFP, 4.6 mm × 250 mm, 4 μm; Mobile phase A: Measure 1 mL of phosphoric acid, add 1000 mL of water, mix well, and adjust the pH to 3.0 with triethylamine. Mobile phase B: Acetonitrile Flow rate: 1.0 mL / min; Column temperature: 40℃; Detection wavelength: 245nm; Injection volume: 20 μL; Elution ratio: The volume ratio of mobile phase A to mobile phase B is 55:45; The structural formula of the pitavastatin calcium starting material is: ; The impurities include: impurity A, impurity B and impurity C; The structure of impurity A is as follows: ; The structure of impurity B is as follows: ; The structure of the impurity C is as follows: .

2. The quantitative analysis method according to claim 1, characterized in that, The preparation methods for the impurity A stock solution, impurity C stock solution, and impurity B stock solution are as follows: Take 5 mg of each of the reference standards for impurity A, impurity C, and impurity B, accurately weigh them, place them in 10 mL volumetric flasks, dissolve them in dimethyl sulfoxide and dilute them to the mark, shake well, and use them as impurity A stock solution, impurity C stock solution, and impurity B stock solution, respectively.

3. The quantitative analysis method according to claim 1, characterized in that, The preparation methods for the test sample derivatization solution, the reference standard derivatization solution, and the 100% spiked test sample derivatization solution in step 1) are as follows: A) Derivatized solution of the test sample Accurately weigh 20 mg of pitavastatin calcium starting material for the test sample and place it in a 5 mL volumetric flask. Add 2 mL of dimethyl sulfoxide to dissolve it, then add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Add dimethyl sulfoxide and dilute to the mark. Shake well to obtain a 0.2 mg / mL derivatized solution of the test sample. B) Reference standard derivatized solution Accurately measure 1 mL of the mixed impurity stock solution and place it in a 5 mL volumetric flask. Add 1 mL of dimethyl sulfoxide, 15 mg of potassium carbonate, and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove from heat and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Dilute to the mark with dimethyl sulfoxide and shake well to obtain a reference standard derivatized solution with a concentration of 0.2 μg / mL. C) 100% spiked test sample derivatized solution Accurately weigh 20 mg of pitavastatin calcium starting material and place it in a 5 mL volumetric flask. Accurately add 1 mL of mixed impurity stock solution and 1 mL of dimethyl sulfoxide to dissolve it. Add 15 mg of potassium carbonate and 15 mg of paraformaldehyde. Stir with a magnetic stirrer and heat at 80 °C for 1 hour. Remove and cool to room temperature. Dilute to the mark with dimethyl sulfoxide and shake well. After standing for 5 minutes, accurately measure 1 mL of the supernatant and place it in a 20 mL volumetric flask. Dilute to the mark with dimethyl sulfoxide and shake well to obtain the final product.

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