Method for separating pitavastatin calcium starting material and impurities thereof

Abstract

The invention relates to the technical field of analytical chemistry, in particular to a method for separating pitavastatin calcium starting materials and impurities thereof, which comprises the following steps: by taking paraformaldehyde as a derivatization reagent, substituting a triphenylphosphine group, amplifying the difference of position isomerism of fluorophenyl, and separating the pitavastatin calcium starting materials from the impurities thereof. The method realizes effective separation of pitavastatin calcium starting materials and impurities thereof, and has the advantages of high separation degree, good repeatability, strong specificity, high sensitivity, good accuracy and strong stability.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for separating a pitavastatin calcium starting material and its impurities. Background Art

[0002] The chemical name of pitavastatin calcium is (+)-bis-{(3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]-3,5-dihydroxy-6-eneheptanoic acid} monocalcium salt. As a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, it is clinically used to treat hyperlipidemia.

[0003] The common preparation of pitavastatin calcium usually uses [[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]methyl]triphenylphosphonium bromide as a raw material, for example: CN103508948A, and the preparation of [[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]methyl]triphenylphosphonium bromide usually uses o-aminobenzoic acid as a raw material, and is obtained by chlorination, Friedel-Crafts alkylation, condensation, reduction, substitution and salt formation with triphenylphosphine. In the Friedel-Crafts alkylation reaction, in addition to the target product at the para position, defluorinated, meta-position and ortho-position isomers are inevitably produced. The defluorinated, meta-position and ortho-position isomers are introduced into the production process of pitavastatin calcium through the witting reaction and directly participate in the subsequent synthesis process. The product polarity is close to that of the pitavastatin calcium intermediate, and it is difficult to remove in the subsequent process, and is finally converted into the defluorinated, ortho-position and meta-position isomer impurities of the pitavastatin calcium finished product.

[0004] The chemical name of the starting material of pitavastatin calcium is: [[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]methyl]triphenylphosphonium 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-quinolyl)methyl]triphenylphosphonium bromide which is a defluorinated impurity of the starting material, impurity B is: [[2-cyclopropyl-4-(3-fluorophenyl)-3-quinolyl]methyl]triphenylphosphonium bromide and impurity C is: [[2-cyclopropyl-4-(2-fluorophenyl)-3-quinolyl]methyl]triphenylphosphonium bromide which is a fluorine position isomer thereof, and the structural formulas of the three are as follows: The presence of the above three impurities will affect the detection of the content of the starting material. Although there are reports on the separation method of the above compounds and their impurities, such as CN115166104B, CN115290791B, etc., the above separation methods have problems such as poor separation and poor accuracy. Therefore, it is urgent to provide a separation method of pitavastatin calcium starting material and its impurities with high separation and good accuracy. Summary of the invention

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

[0007] The present invention is achieved through the following technical solutions:

[0008] The present invention provides a method for separating a pitavastatin calcium starting material and its impurities, characterized in that the separation method adopts a post-derivation liquid chromatography method and comprises the following steps:

[0009] 1). Preparation of test sample derivatization solution, reference sample derivatization solution and 100% spiked test sample derivatization solution:

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

[0011] B) Preparation of reference substance derivatization solution: accurately measure a certain amount of mixed impurity stock solution, place it in a volumetric flask, perform derivatization reaction using derivatization method 1, and then use dimethyl sulfoxide solution and / or diluent to prepare a reference substance derivatization solution with a concentration of about 0.2 μg / mL;

[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, perform derivatization reaction using derivatization method 1, and then use dimethyl sulfoxide solution and / or diluent to 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;

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

[0014] The reaction formula of the derivatization method 1 is:

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

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

[0017] The preparation method of the mixed impurity stock solution is: accurately measure a certain amount of impurity A stock solution, impurity C stock solution and impurity B stock solution respectively, put them in a volumetric bottle, add dimethyl sulfoxide to dilute to the scale, shake well, and obtain;

[0018] Wherein, the structural formula of the pitavastatin calcium starting material is:

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

[0020] The structure of the impurity A is:

[0021] The structure of the impurity B is:

[0022] The structure of the impurity C is:

[0023] Further, as a preferred technical solution of the present invention, the analysis conditions of the liquid chromatography are:

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

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

[0026] Mobile phase B: acetonitrile

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

[0028] Column temperature: 38-42°C;

[0029] Detection wavelength: 245nm;

[0030] Injection volume: 20 μL;

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

[0032] Further, as a preferred technical solution of the present invention, the analysis conditions of the liquid chromatography are:

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

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

[0035] Mobile phase B: acetonitrile

[0036] Flow rate: 1.0 mL / min;

[0037] Column temperature: 40°C;

[0038] Detection wavelength: 245nm;

[0039] Injection volume: 20 μL;

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

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

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

[0043] Further, as a preferred technical solution of the present invention, in the step B), the volume mass ratio of the mixed impurity stock solution: potassium carbonate: paraformaldehyde is: 1 mL: 15 mg: 15 mg;

[0044] Further, as a preferred technical solution of the present invention, in the step C), the volume 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] Further, as a preferred technical solution 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] Further, as a preferred technical solution of the present invention: the preparation of the test sample derivatization solution, the reference sample derivatization solution and the 100% spiked test sample derivatization solution in step 1) is as follows:

[0047] A) Test sample derivatization solution

[0048] Take about 20 mg of pitavastatin calcium starting material test sample, accurately weigh it, place it in a 5 mL volumetric flask, add 2 mL of dimethyl sulfoxide to dissolve it, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to the scale, shake it well, and you can get a test sample derivative solution with a concentration of about 0.2 mg / mL.

[0049] B) Reference substance derivatization solution

[0050] Accurately measure 1 mL of the mixed impurity stock solution, place it in a 5 mL volumetric flask, add 1 mL of dimethyl sulfoxide, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you will get a reference substance derivative solution with a concentration of about 0.2 μg / mL.

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

[0052] Take about 20 mg of pitavastatin calcium starting material test sample, accurately weigh it, 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, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and obtain it.

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

[0054] Further, as a preferred technical solution of the present invention, the preparation method of the mixed impurity stock solution is: accurately measure 1 mL of impurity A stock solution, impurity C stock solution and impurity B stock solution, respectively, put them in a 25 mL volumetric bottle, add dimethyl sulfoxide to dilute to the scale, shake well, and obtain;

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

[0056] Furthermore, as a preferred technical solution 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 adopts liquid chromatography after derivatization, comprising the following steps:

[0057] 1) Derivatization and preparation of solution:

[0058] A) Blank derivatization solution

[0059] Take 2mL of dimethyl sulfoxide, place it in a 5mL volumetric flask, add 15mg of potassium carbonate and 15mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80℃ for 1 hour, take it out and cool to room temperature, dilute to the scale with dimethyl sulfoxide, shake well, let it stand for about 5 minutes, accurately measure 1mL of supernatant, place it in a 20mL volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0060] B) Test sample derivatization solution

[0061] Take about 20 mg of pitavastatin calcium starting material test sample, accurately weigh it, place it in a 5 mL volumetric flask, add 2 mL of dimethyl sulfoxide to dissolve it, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add the diluent to the scale, shake it well, and you will get a test sample derivative solution with a concentration of about 0.2 mg / mL.

[0062] C) Reference substance derivatization solution

[0063] Accurately measure 1 mL of the mixed impurity stock solution, place it in a 5 mL volumetric flask, add 1 mL of dimethyl sulfoxide, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add the diluent to dilute it to the scale, shake it well, and you will get a reference substance derivative solution with a concentration of about 0.2 μg / mL.

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

[0065] Take about 20 mg of the test sample, accurately weigh it, 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 it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add the diluent to dilute it to the scale, shake it well, and you have it.

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

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

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

[0069]

[0070] Furthermore, as a preferred technical solution of the present invention, the present invention also provides an application of a derivative prepared according to the derivatization method 1 of the present invention as a reference substance 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) The present invention adopts a pre-column derivatization method, uses paraformaldehyde as a derivatization reagent, replaces the triphenylphosphine group, amplifies the difference in the positional isomerism of the fluorophenyl group, and achieves effective separation of the pitavastatin calcium starting material and its impurities, and has the advantages of high separation, good repeatability, strong specificity, high sensitivity, good accuracy and strong stability;

[0073] 2) The derivatization method of the present invention is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] 1) Figure 1 It is a specificity overlay diagram of pitavastatin calcium starting material and impurity derivatives;

[0075] 2) Figure 2 is the linear graph of the impurity A derivative;

[0076] 3) Figure 3 is the linear graph of the impurity C derivative;

[0077] 4) Figure 4 is the linear graph of the impurity B derivative;

[0078] 5) Figure 5 It is the liquid chromatography overlay of Comparative Example 1;

[0079] 6) Figure 6 It is the liquid chromatography overlay of Comparative Example 2;

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

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

[0082] 9) Fig. 9 is the mass spectrum of the impurity B derivative;

[0083] 10) Fig.10 This is the mass spectrum of the impurity C derivative. DETAILED DESCRIPTION

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

[0085] Test product information

[0086]

[0087] Reference Material Information

[0088] name source batch number content 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% phosphoric acid aqueous solution (adjust pH to 3.0 with triethylamine)-acetonitrile (55:45)

[0094] Measure 1 mL of phosphoric acid, add it to 1000 mL of water, mix well, and adjust the pH value to 3.0 with triethylamine. Measure 550 mL of the above solution, add 450 mL of acetonitrile, mix well, and you have it. (Or prepare according to the proportion)

[0095] 2) Dilution / blank solution: acetonitrile-water (volume ratio 50:50)

[0096] 3) Test solution

[0097] Take about 10 mg of pitavastatin calcium starting material test sample, accurately weigh it, dissolve it with diluent and make up to the scale to prepare a test sample solution with a concentration of about 0.1 mg / mL.

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

[0099] Take about 5 mg of each of the impurity A, impurity B and impurity C reference substances, accurately weigh them, dissolve them with diluent and make up to the scale to prepare the impurity A, impurity B and impurity C positioning solutions with a concentration of about 0.1 μg / mL.

[0100] 5) Mixed impurity stock solution

[0101] Take about 5 mg of each of the impurity A reference substance, the impurity B reference substance and the impurity C reference substance, accurately weigh them, dissolve them with diluent and make up to the scale to prepare a mixed impurity stock solution with a concentration of about 10 μg / mL.

[0102] 6) 100% spiked test solution

[0103] Take about 10 mg of pitavastatin calcium starting material for test, accurately weigh, and accurately add 1 mL of mixed impurity stock solution, dissolve with diluent and make up to the scale to prepare a spiked test solution with a concentration of 100%.

[0104] 7) Blank derivatization solution

[0105] Take 2mL of dimethyl sulfoxide, place it in a 5mL volumetric flask, add 15mg of potassium carbonate and 15mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80℃ for 1 hour, take it out and cool to room temperature, dilute to the scale with dimethyl sulfoxide, shake well, let it stand for about 5 minutes, accurately measure 1mL of supernatant, place it in a 20mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute to the scale, shake well, and obtain.

[0106] 8) Test sample derivatization solution

[0107] Take about 20 mg of pitavastatin calcium starting material test sample, accurately weigh it, place it in a 5 mL volumetric flask, add 2 mL of dimethyl sulfoxide to dissolve it, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to the scale, shake it well, and you can get a test sample derivative solution with a concentration of about 0.2 mg / mL.

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

[0109] Take about 5 mg of each impurity A, impurity B and impurity C reference substance, accurately weigh them, place them in 10 mL volumetric bottles, add dimethyl sulfoxide to dissolve and dilute to the scale, shake well, as the stock solutions of impurities A, impurity B and impurity C; accurately measure 1 mL of each impurity A, impurity B and impurity C stock solution, place them in 25 mL volumetric bottles, add dimethyl sulfoxide to dilute to the scale, shake well; accurately measure 1 mL, place them in 5 mL volumetric bottles, add dimethyl sulfoxide to 1mL, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde respectively, stir with a magnetic stirrer and heat at 80℃ for 1 hour, take out and cool to room temperature, dilute to the scale with dimethyl sulfoxide respectively, and shake well; after standing for about 5 minutes, accurately measure 1mL of supernatant respectively, put them in 20mL volumetric flasks respectively, add dimethyl sulfoxide and / or diluent to the scale respectively, shake well, and obtain the derivatization location solution of impurity A, impurity B and impurity C with a concentration of about 0.2μg / mL.

[0110] 10) Reference substance derivatization solution

[0111] Accurately measure 1 mL of the mixed impurity stock solution, place it in a 5 mL volumetric flask, add 1 mL of dimethyl sulfoxide, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you will get a reference substance derivative solution with a concentration of about 0.2 μg / mL.

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

[0113] Take about 20 mg of the test sample, accurately weigh it, 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 it, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 ml of the supernatant, place it in a 20 ml volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you have it.

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

[0115] Take about 20 mg of the test sample, accurately weigh it, 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 it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you have it.

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

[0117] Take about 20 mg of the test sample, accurately weigh it, 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 it, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 ml of the supernatant, place it in a 20 ml volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you have it.

[0118] 2. Experimental operation: Take 20 μl of blank derivatization solution, derivatization positioning solution of impurity A, impurity B and impurity C, reference derivatization solution and 100% spiked test derivatization solution and inject them respectively, and record the chromatogram.

[0119] 3. Chromatographic conditions

[0120] Table 1

[0121]

[0122]

[0123] Example 2 Specificity

[0124] Take 1 part of blank derivatization solution prepared in Example 1, 1 part of reference derivatization solution, 1 part of derivatization location solution of impurity A, 1 part of derivatization location solution of impurity B, 1 part of derivatization location solution of impurity C, 1 part of test sample derivatization solution, and 1 part of 100% spiked test sample derivatization solution. Except for 5 injections of reference sample derivatization solution, 1 injection of each of the other solutions is performed. At the end of the sequence, 1 injection of reference sample derivatization solution is performed. The specific chromatographic conditions are shown in Table 1. Isocratic elution is used and the chromatogram is recorded. The results are shown in Tables 2 and 3. Figure 1 .

[0125] Table 2 Results of impurity specificity test system suitability

[0126]

[0127] Table 3 Impurity specificity test results

[0128]

[0129]

Note

[0130] The test results show that the blank derivative solution has no interference with the detection. In the reference derivative solution, the minimum separation 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 5 needles and all reference derivative solutions is 1.8%. In the 100% spiked test solution, the retention time of each target impurity derivative peak is consistent with the retention time of the target impurity derivative peak in each positioning solution; the minimum separation between each target impurity derivative peak and the adjacent peak, and between the main component derivative peak and the adjacent peak is 3.3; the recovery rate of each target impurity detection result is between 80% and 120%. This method has good specificity.

[0131] Example 3 Linear

[0132] The concentration of the quantitative limit is taken as the lowest point of the linear range, and the mixed impurity stock solution is diluted with dimethyl sulfoxide in an appropriate amount, 15 mg of potassium carbonate and 15 mg of paraformaldehyde are added, stirred with a magnetic stirrer and heated at 80°C for 1 hour, taken out and cooled to room temperature, diluted to the scale with dimethyl sulfoxide, and shaken well; after standing for about 5 minutes, 1 mL of the supernatant is accurately measured, placed in a 20 mL volumetric flask, and the diluent is added to dilute to the scale, shaken well, and a series of solutions equivalent to 30% to 200% of the limit concentration are prepared. As linear solutions, they are injected into the liquid chromatograph respectively, and eluted isocratically. The chromatographic conditions are the same as those in Table 1, and the spectrum is recorded. The relationship between the different concentrations of each target impurity and the peak area was examined together with the quantitative limit solution. The least square method was used for linear regression. The correlation coefficient r should not be less than 0.998, the ratio of the Y-axis intercept to the 100% response value should not exceed 25%, the response factor (A / C) RSD should be ≤10%, and the residual sum of squares should be reported; a residual graph should be attached, and the residual points should be scattered 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 derivatives

[0134]

[0135]

[0136] Table 5 Linearity test results of impurity C derivatives

[0137]

[0138] Table 6 Linearity test results of impurity B derivatives

[0139]

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

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

[0142] Limit of Quantitation

[0143] Take the reference substance derivative solution for liquid chromatography injection, isocratic elution, and the chromatographic conditions are the same as those in Table 1, so that the main peak height is 10 times the noise peak height. This concentration is used as the quantitative concentration, and its quantitative limit is calculated. The signal-to-noise ratio of the quantitative limit solution should not be less than 10; repeat the injection 6 times, and the RSD of the peak area should be ≤10%. The quantitative limit concentration shall not be greater than 30% of the limit (0.06μg / mL). The results are shown in Table 7.

[0144] Table 7 Results of quantitation limit test of impurity derivatives

[0145]

[0146]

[0147] The experimental results show that this method has good sensitivity and can effectively and quantitatively detect the target impurities.

[0148] Detection limit

[0149] The reference substance derivatization solution was injected into the liquid chromatography and eluted isocratically. The chromatographic conditions were the same as those in Table 1, so that the main peak height was 3 times the noise peak height. This concentration was taken as the minimum detection concentration, and the minimum detection amount was calculated. The results are shown in Table 8.

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

[0151]

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

[0153] Example 5 Precision

[0154] Repeatability

[0155] Analyst 1 takes 1 blank derivatization solution, 1 reference derivatization solution, and 6 100% spiked test sample derivatization solutions. Except for 5 injections of reference sample derivatization solution, the other solutions are injected with 1 injection each. At the end of the sequence, 1 injection is returned to the reference sample solution. The chromatographic conditions are the same as those in Table 1, and the chromatogram is recorded. The RSD or range of the detected amount of each target impurity in the 6 100% spiked test sample derivatization solutions should meet the requirements of the following table:

[0156] Test results Relative standard deviation (RSD) / range 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 System suitability results of impurity derivative repeatability test

[0159]

[0160] Table 10 Impurity derivative repeatability test results

[0161]

[0162]

[0163] The test results show that the blank derivatization solution has no interference with the detection. In the reference derivatization solution, the minimum separation between the peaks of each target impurity derivative is 5.6; the maximum RSD of the peak area of ​​each target impurity derivative in 5 injections and all reference derivatization solutions is 1.8%. In the 6 100% spiked test derivatization solutions, the RSD of the detected amount of each target impurity meets the verification requirements, and the repeatability of this method is good.

[0164] Example 6 Intermediate Precision

[0165] Analyst 2 uses a different LC instrument on a different date and performs the repeatability test. Calculate the detection amount and RSD of each target impurity in the 6 100% spiked test sample derivative solutions and compare them with the repeatability results. The RSD or range of each target impurity detection amount in the 6 100% spiked test sample derivative solutions of Analyst 2 and the RSD or range of each target impurity detection amount in the 12 100% spiked test sample derivative solutions of 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 System suitability results for intermediate precision test of impurity derivatives

[0169]

[0170] Table 12 Results of intermediate precision test of impurity derivatives

[0171]

[0172] The test results show that the blank derivatization solution has no interference with the detection. In the reference derivatization solution, the minimum separation 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 5 injections and all reference derivatization solutions is 0.6%. In the 6 100% spiked test derivatization solutions of analyst 2, the RSD of the detected amount of each target impurity meets the verification requirements, and the intermediate precision of this method is good.

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

[0174] Example 7 Accuracy

[0175] Take 1 blank derivatization solution, 1 reference derivatization solution, 1 test derivatization solution, 3 50% spiked test derivatization solutions, 3 100% spiked test derivatization solutions, and 3 150% spiked test derivatization solutions. Except for the reference derivatization solution, which was injected 5 times, the other solutions were injected 1 time each, and the reference derivatization solution was injected 1 time at the end of the sequence. According to the chromatographic conditions in Table 1, chromatographic injection was performed and the chromatogram was recorded. The results are shown in Tables 13 to 16.

[0176] Table 13 System suitability results of impurity derivative repeatability test

[0177]

[0178] Table 14 Accuracy test results of impurity A derivatives

[0179]

[0180] Table 15 Accuracy test results of impurity C derivatives

[0181]

[0182]

[0183] Table 16 Accuracy test results of impurity B derivatives

[0184]

[0185] The test results show that the blank derivative solution has no interference with the detection. In the reference derivative solution, the minimum separation between the peaks of each target impurity derivative is 6.4; the maximum RSD of the peak area of ​​each target impurity derivative in 5 injections and all reference derivative solutions is 0.6%. In the 9 spiked test solutions, the recovery rate of each target impurity derivative is within the range of 90% to 110%; the RSD of the recovery rate of each target impurity derivative meets the verification requirements, and the accuracy of this method is good.

[0186] Example 8 Solution Stability

[0187] Prepare 1 blank derivatization solution, 1 reference derivatization solution, and 100% spiked test sample derivatization solution. Place the reference derivatization solution and 100% spiked test sample derivatization solution at room temperature (10°C to 30°C) without light, and inject samples at different time points according to the chromatographic conditions in Table 1, and record the chromatogram. Relative to the stability solution at 0 hours, the absolute value of the change rate of the peak area of ​​each target impurity derivative at each stability time point in the reference derivatization solution should be ≤10.0%; the absolute value of the change rate / absolute value of the detected amount of each target impurity at each stability time point in the 100% spiked test sample derivatization solution should meet the requirements of the following table:

[0188] Impurity derivative content determination value Absolute value of detection rate of change / absolute value of change Measured value < 0.03% (or reporting limit) Absolute value of change ≤ 0.01% 0.03% (or reporting limit) ≤ measured value < 0.10% Absolute value of change ≤ 0.02% 0.10%≤measured value<0.2% Absolute value of change rate ≤30% 0.2%≤measured value<0.5% Absolute value of change rate ≤ 20% 0.5%≤measured value<2.0% Absolute value of change rate ≤ 10% 2.0%≤measured value Absolute value of change rate ≤5%

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

[0190] Table 17 Results of stability test of reference substance derivative solution

[0191]

[0192]

[0193]

Note

[0194] Table 18100% spiked test sample derivative solution stability test results

[0195]

[0196]

Note

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

[0198] Example 9 Durability

[0199] The influence of changing the flow rate, column temperature and derivatization time on the test results was investigated. The change parameters are shown in the table below:

[0200] Chromatographic conditions / derivatization conditions Specified value Range of change Flow rate 1.0mL / min 0.8mL / min and 1.2mL / min Column temperature 40℃ 38°C and 42°C Derivative Time 1 hour (60 minutes) 50 minutes and 70 minutes

[0201] Each time one of the conditions was changed and the other conditions were kept unchanged, 1 blank derivatization solution, 1 reference derivatization solution, and 1 100% spiked test sample derivatization solution were prepared. Except for the reference derivatization solution, which was injected 5 times, the other solutions were injected 1 time each, and 1 time was injected back to the reference derivatization solution at the end of the sequence, and the chromatogram was recorded. The results are shown in Tables 19 and 20.

[0202] Table 19 Impurity derivatives durability test system suitability results

[0203]

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

[0205] Table 20 Impurity Derivatives Impurity Durability Test Results

[0206]

[0207] [Note] The durability of standard chromatographic conditions (flow rate 1.0 mL / min, column temperature 40°C, derivatization time 60 min) refers to the measurement result of the first 100% spiked test solution under the intermediate precision item.

[0208] The experimental results show that when there are slight changes in the chromatographic conditions and derivatization conditions (flow rate 0.8mL / min~1.2mL / min, column temperature 38℃~42℃, derivatization time 50min~70min), the RSD of the detection amount of each target impurity in the 100% spiked test solution meets the validation requirements, and this method has good durability.

[0209] Example 10 Sample Testing

[0210] Prepare 1 blank derivatization solution, 1 reference derivatization solution, and 4 batches of samples (batch numbers D2202091, N8.2-C231101, N8.2-C231102, and N8.2-C231103), and prepare 1 test sample derivatization solution for each batch of samples. Perform liquid chromatography analysis according to the chromatographic conditions in Table 1, and record the chromatogram. The results are shown in Tables 21 and 22.

[0211] Table 21 Results of the suitability test of the impurity derivative sample detection system

[0212]

[0213] Table 22 Impurity derivative sample test results

[0214]

[0215] Conclusion: The blank derivatization solution had no interference with the detection. The minimum separation between the peaks of the target impurity derivatives in the reference derivatization solution was 5.6; the maximum RSD of the peak area of ​​the target impurity derivatives in the 5 injections and all reference derivatization solutions was 1.8%. The impurities detected in the 4 batches of samples all met the acceptable standards.

[0216] Example 11 Structural Identification of Test Sample Derivatives and Impurity Derivatives:

[0217] 11.1 Instrument / Material Information:

[0218] Column No.: QCL400 QCL295

[0219] HPLC Model: Waters H-Class No.: QC-OB03-006 Calibration Expiration Date: 2025.07.17

[0220] LC-MS Model: Agilent 6470 Number: QC-LCMS-001 Calibration Expiration Date: 2025.04.17

[0221] 11.1.1 Chromatographic conditions:

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

[0223] Mobile phase A: 0.1% formic acid in water

[0224] Mobile phase B: acetonitrile

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

[0226] Flow rate: 0.5ml / min

[0227] Column temperature: 40°C

[0228] Injection volume: 20 μL

[0229] Run time: 15 minutes

[0230] 11.1.2 Mass spectrometry conditions:

[0231] Ion source: APCI, positive mode

[0232] Scan mode: MS2 Scan

[0233] Data collection time period: 3 to 15 minutes

[0234] 11.2 Solution preparation:

[0235] Blank solution / dilution: 50% acetonitrile-water solution

[0236] 11.2.1 Test product derivative solution: Take about 20 mg of pitavastatin calcium starting material test product, weigh accurately, place in a 5 ml volumetric flask, add 2 ml of dimethyl sulfoxide to dissolve, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take out and cool to room temperature, dilute to scale with dimethyl sulfoxide, shake well; let stand for about 5 minutes. Measure 0.5 ml of the above solution, place in a colorimetric tube, add 3 ml of ethyl acetate, add 10 ml of ultrapure water, shake to separate, take 0.5 ml of the upper layer solution, place in a 10 ml volumetric flask, evacuate to evaporate the solvent, add 2 ml of diluent to dissolve the remaining residue, and obtain.

[0237] Dilution of test sample derivative: Measure 0.5 ml of the above test sample derivative solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, shake well, and the solution is ready. The above prepared dilution of test sample derivative is subjected to LC-MS analysis. The LC-MS results are as follows: Figure 7 shown.

[0238] 11.2.2 Impurity A derivative solution: Take about 5 mg of the impurity A reference substance, place it in a 10 ml volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the mark, shake well, and use it as 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, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and let it cool to room temperature, dilute to the mark with dimethyl sulfoxide, shake well; let it stand for about 5 minutes. Measure 0.5 ml of the above solution, place it in a colorimetric tube, add 3 ml of ethyl acetate, add 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, evacuate the vacuum to evaporate the solvent, add 2 ml of diluent to dissolve the remaining residue, and you have it.

[0239] Impurity A derivative dilution: Take 0.5 ml of the above impurity A derivative solution, place it in a 10 ml volumetric flask, add dilution to dilute to the mark, shake well, and the result is obtained. The above prepared impurity A derivative dilution is subjected to LC-MS analysis. The LC-MS results are as follows: Figure 8 shown.

[0240] 11.2.3 Solution of impurity B derivative: Take about 5 mg of impurity B reference substance, place it in a 10 ml volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the mark, shake well, and use it as 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, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and let it cool to room temperature, dilute to the mark with dimethyl sulfoxide, shake well; let it stand for about 5 minutes. Measure 0.5 ml of the above solution, place it in a colorimetric tube, add 3 ml of ethyl acetate, add 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, evacuate the vacuum to evaporate the solvent, add 2 ml of diluent to dissolve the remaining residue, and you have it.

[0241] Impurity B derivative dilution: Take 0.5 ml of the above impurity B derivative solution, place it in a 10 ml volumetric flask, add dilution to dilute to the mark, shake well, and the result is obtained. The above prepared impurity B derivative dilution is subjected to LC-MS analysis. The LC-MS results are as follows: Fig. 9 shown.

[0242] 11.2.4 Impurity C derivative solution: Take about 5 mg of the impurity C reference substance, place it in a 10 ml volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the mark, shake well, and use it as 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, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and let it cool to room temperature, dilute to the mark with dimethyl sulfoxide, shake well; let it stand for about 5 minutes. Measure 0.5 ml of the above solution, place it in a colorimetric tube, add 3 ml of ethyl acetate, add 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, evacuate the vacuum to evaporate the solvent, add 2 ml of diluent to dissolve the remaining residue, and you have it.

[0243] Impurity C derivative dilution: Take 0.5 ml of the above impurity C derivative solution, place it in a 10 ml volumetric flask, add dilution to dilute to the mark, shake well, and the result is obtained. The above prepared impurity C derivative dilution is subjected to LC-MS analysis. The LC-MS results are as follows: Fig.10 shown.

[0244] Comparative Example 1

[0245] Sample preparation: Use the diluent as the 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] Test operation: Take 20 μL of blank solution, test solution, reference solution and 100% spiked test solution, analyze according to the chromatographic conditions in Table 1, and record the chromatogram. Figure 5.

[0247] according to Figure 5 It can be seen from the results that the impurities can be completely separated from each other, but impurity B cannot be completely separated from the main peak, which indicates that without derivatization, it is difficult to separate the pitavastatin calcium starting material test sample from the impurities even under the chromatographic conditions of the present invention.

[0248] Comparative Example 2

[0249] Derivative method 2:

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

[0251] The specific steps of derivatization method 2 are: 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 acetone, stir with a magnetic stirrer and heat at 80°C for 1 hour, take out and cool to room temperature, dilute to the scale with dimethyl sulfoxide, and shake well.

[0252] Sample preparation:

[0253] 1) Blank derivatization solution 2

[0254] Take 2mL of dimethyl sulfoxide, place it in a 5mL volumetric flask, add 15mg of potassium carbonate and 60μL of acetone, stir with a magnetic stirrer and heat at 80℃ for 5 hours, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, shake it well, let it stand for about 5 minutes, accurately measure 1mL of the supernatant, place it in a 20mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you have it.

[0255] 2) Test sample derivatization solution 2

[0256] Take about 20 mg of pitavastatin calcium starting material test sample, accurately weigh it, place it in a 5 mL volumetric flask, add 2 mL of dimethyl sulfoxide to dissolve it, add 15 mg of potassium carbonate and 60 μL of acetone, stir it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to the scale, shake it well, and you will get a test sample derivative solution with a concentration of about 0.2 mg / mL.

[0257] 3) Derivatization location solution 2 of impurity A, impurity B and impurity C

[0258] Take about 5 mg of each impurity A, impurity B and impurity C reference substance, accurately weigh them, place them in 10 mL volumetric bottles, add dimethyl sulfoxide to dissolve and dilute to the scale, shake well, as the stock solutions of impurities A, impurity B and impurity C; accurately measure 1 mL of each impurity A, impurity B and impurity C stock solution, place them in 25 mL volumetric bottles, add dimethyl sulfoxide to dilute to the scale, shake well; accurately measure 1 mL, place them in 5 mL volumetric bottles, add dimethyl sulfoxide to 1mL, add 15mg of potassium carbonate and 60μL of acetone respectively, stir with a magnetic stirrer and heat at 80℃ for 5 hours, take out and cool to room temperature, dilute to the scale with dimethyl sulfoxide respectively, and shake well; after standing for about 5 minutes, accurately measure 1mL of supernatant respectively, put them in 20mL volumetric flasks respectively, add dimethyl sulfoxide and / or diluent to the scale respectively, shake well, and obtain the derivatization location solution 2 of impurity A, impurity B and impurity C with a concentration of about 0.2μg / mL.

[0259] 4) Reference substance derivatization solution 2

[0260] Accurately measure 1 mL of the mixed impurity stock solution, place it in a 5 mL volumetric flask, add 1 mL of dimethyl sulfoxide, add 15 mg of potassium carbonate and 60 μL of acetone, stir with a magnetic stirrer and heat at 80°C for 5 hours, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you will get a reference substance derivative solution 2 with a concentration of about 0.2 μg / mL.

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

[0262] Take about 20 mg of the test sample, accurately weigh it, 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 60 μL of acetone, stir it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you have it.

[0263] 6) Experimental operation: Take 20 μL of blank derivatization solution 2, test sample derivatization solution 2, impurity A, impurity B and impurity C derivatization solution 2, reference sample derivatization solution 2 and 100% spiked test sample derivatization solution 2, respectively, and analyze according to the chromatographic conditions in Table 1, and record the chromatogram. The results are shown in Figure 6 .

[0264] according to Figure 6 It can be seen from the results that by using the above-mentioned derivatization method 2, the impurities and the main component can be completely separated, but more 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. For ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for separating a pitavastatin calcium starting material and its impurities, characterized in that: The separation method adopts liquid chromatography after derivatization, comprising the following steps: 1) Preparation of test sample derivatization solution, reference sample derivatization solution and 100% spiked test sample derivatization solution: A) Preparation of the test sample derivatization solution: Take a certain weight of the pitavastatin calcium starting material test sample, place it in a volumetric flask, perform derivatization reaction using derivatization method 1, and then use dimethyl sulfoxide solution and / or diluent to prepare a test sample derivatization solution with a concentration of about 0.2 mg / mL; B) Preparation of reference substance derivatization solution: accurately measure a certain amount of mixed impurity stock solution, place it in a volumetric flask, perform derivatization reaction using derivatization method 1, and then use dimethyl sulfoxide solution and / or diluent to prepare a reference substance derivatization solution with a concentration of about 0.2 μg / mL; C) Preparation of 100% spiked test sample derivatization solution: a certain weight of pitavastatin calcium starting material test sample and a certain volume of mixed impurity stock solution are taken for derivatization reaction using derivatization method 1, and then a 100% spiked test sample derivatization solution containing 0.2 mg pitavastatin calcium starting material test sample, 0.2 μg impurity A derivative, 0.2 μg impurity C derivative and 0.2 μg impurity B derivative per 1 mL is prepared with dimethyl sulfoxide solution and / or diluent; 2) taking 20 μL of the solution prepared in step 1) respectively, injecting them into a liquid chromatography system, and performing qualitative or quantitative analysis at a wavelength of 245 nm; Wherein, the reaction formula of the derivatization method 1 is: The R is selected from: H or F; The specific steps of the derivatization method 1 are: accurately weigh a certain weight of pitavastatin calcium starting material, impurities A, B and / or C, place them in a volumetric bottle, add a certain amount of dimethyl sulfoxide to dissolve, add potassium carbonate and paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take out and cool to room temperature, dilute to scale with dimethyl sulfoxide, and shake well; The preparation method of the mixed impurity stock solution is: accurately measure a certain amount of impurity A stock solution, impurity C stock solution and impurity B stock solution respectively, put them in a volumetric bottle, add dimethyl sulfoxide to dilute to the scale, shake well, and obtain; The structural formula of the pitavastatin calcium starting material is: The impurities include: impurity A, impurity B and impurity C; The structure of the impurity A is: The structure of the impurity B is: The structure of the impurity C is:

2. The separation method according to claim 1, characterized in that The analysis conditions of the liquid chromatography are: Column: Agilent InfinityLab Poroshell 120PFP, 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 value to 3.0 with triethylamine; Mobile phase B: acetonitrile Flow rate: 0.8-1.2mL / min; Column temperature: 38-42°C; Detection wavelength: 245nm; Injection volume: 20 μL; Elution ratio: The volume ratio of mobile phase A: mobile phase B is 55:

45.

3. The separation method according to claim 2, characterized in that The analysis conditions of the liquid chromatography are: Column: Agilent InfinityLab Poroshell 120PFP, 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 value to 3.0 with triethylamine; Mobile phase B: acetonitrile Flow rate: 1.0 mL / min; Column temperature: 40°C; Detection wavelength: 245nm; Injection volume: 20 μL; Elution ratio: The volume ratio of mobile phase A: mobile phase B is 55:

45.

4. The separation method according to claim 1, characterized in that The derivatization reaction time in step 1) is 50 min-70 min.

5. The separation method according to claim 1, characterized in that In the step A), the mass ratio of pitavastatin calcium starting material: potassium carbonate: paraformaldehyde is 4:3:3; In the step B), the volume mass ratio of the mixed impurity stock solution: potassium carbonate: paraformaldehyde is: 1 mL: 15 mg: 15 mg; In the step C), the volume mass ratio of the mixed impurity stock solution: pitavastatin calcium starting material: potassium carbonate: paraformaldehyde is: 1 mL: 20 mg: 15 mg: 15 mg.

6. The separation method according to claim 1, characterized in that 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.

7. The separation method according to claim 4, characterized in that The preparation methods of the impurity A stock solution, the impurity C stock solution and the impurity B stock solution are as follows: about 5 mg of each of the impurity A, impurity C and impurity B reference substances are taken, accurately weighed, placed in 10 mL volumetric bottles, respectively, and dimethyl sulfoxide is added to dissolve and dilute to the scale, shaken well, and used as the impurity A stock solution, impurity C stock solution and impurity B stock solution, respectively.

8. The separation method according to claim 1, characterized in that The preparation method of the test sample derivatization solution, the reference sample derivatization solution and the 100% spiked test sample derivatization solution in step 1) is as follows: A) Test sample derivatization solution Take about 20 mg of the starting material of pitavastatin calcium for testing, accurately weigh it, put it in a 5 mL volumetric flask, add 2 mL of dimethyl sulfoxide to dissolve it, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir it with a magnetic stirrer and heat it at 80°C for 1 hour, take it out and let it cool to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, put it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to the scale, shake it well, and obtain a test sample derivative solution with a concentration of about 0.2 mg / mL; B) Reference substance derivatization solution Accurately measure 1 mL of the mixed impurity stock solution, place it in a 5 mL volumetric flask, add 1 mL of dimethyl sulfoxide, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of the supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and you will get a reference substance derivative solution with a concentration of about 0.2 μg / mL; C) 100% spiked test sample derivatization solution Take about 20 mg of pitavastatin calcium starting material test sample, accurately weigh it, 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, add 15 mg of potassium carbonate and 15 mg of paraformaldehyde, stir with a magnetic stirrer and heat at 80°C for 1 hour, take it out and cool it to room temperature, dilute it to the scale with dimethyl sulfoxide, and shake it well; after standing for about 5 minutes, accurately measure 1 mL of supernatant, place it in a 20 mL volumetric flask, add dimethyl sulfoxide and / or diluent to dilute it to the scale, shake it well, and obtain it.

9. A derivative prepared by the derivatization method 1 according to claim 1, characterized in that: The derivative is selected from the following structures:

10. Use of the derivative according to claim 9 as a reference substance in the separation and detection of pitavastatin calcium starting material and its impurities.

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