WO2021217887A1 - Testing method for potential mutagenic impurities in pitavastatin calcium tablet - Google Patents

Abstract

A testing method for potential mutagenic impurities in a pitavastatin calcium tablet. After a solvent is used to dissolve a sample to be tested, high performance liquid chromatography is utilized to perform testing, where chromatography conditions are as follows: mobile phase: an organic acid salt buffer solution and acetonitrile; flow rate: 1.0mL / min; column temperature: 25-40 ℃; chromatography column: octadecyl silane bonded silica is used as a filler; injection volume: 50 μL; testing wavelength: 270-280 nm; elution mode: gradient elution; and the potential mutagenic impurities are impurity 1 and impurity 2. The present method can achieve effective separation of pitavastatin calcium, impurity 1, and impurity 2; said method can also prevent another related impurity of pitavastatin calcium from interfering with testing for impurity 1 and impurity 2, and can accurately measure the potential mutagenic impurity 1 and impurity 2 contents in a pitavastatin calcium tablet, effectively monitoring pitavastatin calcium tablet pharmaceutical quality, and increasing medication safety.

Description

Detection method of potential mutagenic impurities in pitavastatin calcium tablets Technical field

The invention relates to the field of drug quality detection, in particular to a detection method of potential mutagenic impurities in pitavastatin calcium tablets.

Background technique

As HMG-CoA reductase inhibitors, statins are widely used for lowering blood lipids and cholesterol, etc., while pitavastatin is known as "super statin", pitavastatin calcium tablets are the latest generation of hypercholesterolemia treatment drugs in the world today Compared with similar products, it has the advantages of small dosage, fewer side effects, and definite curative effect. According to reports, pitavastatin calcium has the effect of significantly lowering low-density lipoprotein cholesterol, and the effect is stronger than several other statin drugs, and the drug is effective for adolescent patients and has a wide range of clinical value.

Pitavastatin calcium is easily degraded after being irradiated with light and produces a variety of impurities, including a variety of potentially mutagenic impurities with warning structures. Impurity 1 and Impurity 2 (see Table 1 for specific structures) are two of them. The above-mentioned impurities in medicines can easily affect the quality and safety of medicines and may cause adverse reactions. According to the requirements of relevant guidelines, they must be studied and controlled.

Table 1 Structure and chemical name of impurity 1, impurity 2

At present, in the pharmacopoeias at home and abroad, except for Pitavastatin calcium tablets included in the Japanese Pharmacopoeia, no other pharmacopoeias include it. After testing, the related substance detection method contained in JP17 Pitavastatin calcium tablets can separate and detect a variety of impurities, but cannot detect the content of impurity 1 and impurity 2. The characteristic peaks of impurity 1 and impurity 2 and other impurity peaks during the detection process Can not be separated, interfere with the detection of impurity 1 and impurity 2.

Summary of the invention

In view of the above shortcomings, the purpose of the present invention is to provide a method for detecting potential mutagenic impurities in pitavastatin calcium tablets, which can effectively separate pitavastatin calcium, impurity 1, and impurity 2, and accurately detect pitavastatin The content of potential mutagenic impurity 1 and impurity 2 of calcium tablets, simple operation, easy to control, high sensitivity, good specificity, system applicability, solution stability and durability of chromatographic conditions, provide an effective way for monitoring product quality The detection method further guarantees the safety of the product.

In the method for detecting potential mutagenic impurities in pitavastatin calcium tablets of the present invention, the sample to be tested is dissolved in a solvent and then detected by high performance liquid chromatography, and the chromatographic conditions are as follows:

Mobile phase: organic acid salt buffer and acetonitrile;

Flow rate: 1.0mL/min;

Column temperature: 25-40°C, preferably 30°C;

Chromatographic column: use octadecyl silane bonded silica gel as filler;

Injection volume: 50μL;

Detection wavelength: 270～280nm, preferably 275nm;

Elution method: gradient elution;

Among them, the potential mutagenic impurities are impurity 1 and impurity 2, and the structure of impurity 1 is:

The structure of impurity 2 is:

in:

The detection is carried out by high performance liquid chromatography, a reversed-phase high performance liquid chromatography system is used, and the gradient elution procedure is as follows:

Time (min)	Organic acid salt buffer (%)	Acetonitrile (%)
0	60	40
5	60	40
40	35	65
42	10	90
50	10	90
51	60	40

The organic acid salt buffer is an acetate buffer with a pH of 3.4-3.6.

The solvent is acetonitrile-water solution, and the volume content of acetonitrile is 40-60%.

The method for detecting potential mutagenic impurities in pitavastatin calcium tablets of the present invention specifically includes the following steps:

(1) Preparation of sample solution;

(2) Preparation of system suitability solution;

(3) Preparation of impurity reference substance solution;

(4) Detection method: adopt high performance liquid chromatography, use gradient elution procedure for impurity separation, and control impurity content by limit method.

Among them, the preferred technical solutions are as follows:

In step (1), the method for preparing the sample solution is: take a sample equivalent to 20 mg of pitavastatin calcium, add a solvent, dissolve by ultrasonic, dilute to a constant volume of 40 mL, centrifuge or stand, and take the supernatant to obtain the sample Solution. Membrane filtration cannot be used in this step, and it must be centrifuged or allowed to stand still.

In step (2), the preparation method of the system suitability solution is: weigh pitavastatin calcium reference substance, pitavastatin calcium impurity 1 reference substance, and pitavastatin calcium impurity 2 reference substance, dissolve and dilute with a solvent to prepare A mixed solution containing pitavastatin calcium 0.5mg/mL, impurity 1 0.15μg/mL, and impurity 2 0.15μg/mL is a system suitability solution.

In step (3), the preparation method of the impurity reference substance solution is as follows: Weigh pitavastatin calcium impurity 1 reference substance and impurity 2 reference substance, dissolve and dilute it with a solvent, and prepare it as containing impurity 1 0.15 μg/mL, impurity 2 0.15 The mixed solution of μg/mL is the impurity reference solution.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. The method of the present invention can realize the effective separation of pitavastatin calcium, impurity 1, and impurity 2; at the same time, it avoids the interference of pitavastatin calcium and other related impurities on the detection of impurity 1, impurity 2, and can accurately detect horses. The content of potential mutagenic impurity 1 and impurity 2 of vastatin calcium tablets can effectively monitor the drug quality of pitavastatin calcium tablets and improve the safety of medication.

2. The method for detecting potential mutagenic impurities in pitavastatin calcium tablets of the present invention has simple operation, easy control, high sensitivity, good specificity, system applicability, solution stability and durability of chromatographic conditions. Monitoring product quality provides an effective detection method to further ensure product safety.

Description of the drawings

Figure 1 is a chromatogram of the system suitability solution;

Figure 2 is a chromatogram of the blank excipient of pitavastatin calcium tablets;

Figure 3 is a chromatogram of the test solution of pitavastatin calcium tablets;

Figure 4 is a chromatogram of the impurity reference solution.

Detailed ways

In order to make the purpose, technical solutions, and effects of the present application clearer and clearer, the present invention will be further described below in conjunction with embodiments. It is only used to illustrate the present invention, but not to limit the scope of the present invention.

Impurity 1 and Impurity 2 can be produced by the degradation of pitavastatin calcium by light. The structure of the two impurities contains warning structure fragments of "polycyclic aromatic hydrocarbons". According to the requirements of ICH M7 and other relevant guidelines, they should be studied and controlled.

The high-performance liquid chromatography used in the following examples, without special instructions, is carried out under the following conditions: the chromatographic column uses octadecylsilane bonded silica gel as the filler, the specification is 250×4.6mm, 5μm, Shimadzu ODS-3 Series; Column temperature: 30℃; Flow rate: 1.0mL/min; UV detector with detection wavelength of 275nm; Injection volume: 50μL.

Wherein, the mobile phase of the high-performance liquid chromatography method contains: mobile phase A, which is a 0.01M acetate buffer, with a pH of 3.5; mobile phase B, which is acetonitrile; and the following procedures are used for gradient elution:

Time (min)	Mobile phase A (%)	Mobile phase B (%)
0	60	40
5	60	40
40	35	65
42	10	90
50	10	90
51	60	40

The preparation method of mobile phase A is as follows: weigh 0.6 g of acetic acid, dilute to 1 L with deionized water, and adjust the pH value to 3.5 with sodium acetate solution.

Tests have proved that the organic filter membrane has the effect of adsorbing impurity 2 and will reduce the chromatographic peak area. Therefore, centrifugation or standing should be used for sample processing, instead of filter membrane filtration.

Preparation of impurity reference solution: Weigh 5.0 mg of impurity 1, impurity 2 and place in a 100mL measuring flask, add solvent to dissolve and dilute to the mark, shake well, as the impurity reference mother liquor; take 3mL of impurity reference mother liquor, and place 100mL volume Dilute the bottle with a solvent to the mark and shake it up as the impurity reference substance stock solution; take 2 mL of the impurity reference substance stock solution, put in a 20 mL measuring flask, dilute to the mark with a solvent, and shake it to obtain the impurity reference substance solution.

Preparation of sample solution: Take 20 1mg pitavastatin calcium tablets, place in a 40mL measuring flask, add solvent to about 2/3 of the volume of the measuring flask, sonicate for 10min, take out, dilute to the mark with solvent, shake well, centrifuge, Take the supernatant as the sample solution.

System suitability solution preparation: Weigh 20.0 mg of pitavastatin calcium reference substance, put it in a 40mL measuring flask, add a proper amount of solvent to ultrasound to dissolve, add 4mL of impurity reference substance stock solution, dilute to the mark with solvent, shake well, and get it.

Preparation of mixed sample solution: Take 20 1mg pitavastatin calcium tablets, put in a 40mL measuring flask, add solvent to about 2/3 of the volume of the measuring flask, sonicate for 10min, take it out, add 4mL of impurity reference substance stock solution, use solvent Dilute to the mark, shake well, centrifuge, and take the supernatant to get.

Example 1

Good specificity:

It has been verified that (1) the solvent and blank excipients do not interfere with the detection of the main peak of pitavastatin calcium, impurity 1 peak and impurity 2 peak; (2) the separation between the main peak and adjacent impurities and between adjacent impurities is not less than 1.5; (3) System suitability The retention time and resolution of each peak in the solution are shown in Table 2.

Table 2 System suitability solution test results

Example 2

Good system applicability:

The solvent does not interfere with the detection of each peak. The number of theoretical plates for the main peak is 15977; the number of theoretical plates for impurity 1 is 45800, and the tailing factor is 1.19; the number of theoretical plates for impurity 2 is 116945, and the tailing factor is 1.05; 5 consecutive injections of the impurity reference solution, impurity 1 and The RSD of impurity 2 peak area is less than 1%. The results show that the system is good.

Example 3

Good sensitivity:

Investigate the detection limit of impurities. Take an appropriate amount of impurity 1, impurity 2 reference solution and gradually dilute it with a solvent. The sample concentration corresponding to the signal-to-noise ratio of about 3:1 or 2:1 is the detection limit. The test results are shown in Table 3.

Table 3 Results of detection limit of impurity 1, impurity 2

Impurity	Concentration (μg/mL)	Peak area	Signal to noise ratio S/N
Impurity 1	0.0068	1683	3.34
Impurity 2	0.0026	813	3.15

Example 4

Good stability:

After testing, the sample used does not contain impurity 1 and impurity 2. In order to better examine the stability of the sample, a mixed sample solution with impurity reference substance was added instead of the sample solution for testing. Take the impurity reference solution and the mixed sample solution, and test them at different times. The test results are shown in Table 4 and Table 5. Within 36 hours of the impurity reference solution and the mixed sample solution, the RSD of each chromatographic peak area is less than 5%, and the stability is good.

Table 4 Stability test results of mixed sample solution

Table 5 Stability test results of impurity reference substance solution

Example 5

Good durability:

By making small changes to each condition in the chromatographic conditions, the degree of influence on the detection results is tested. In the implementation test, the column temperature of the chromatographic column was ±5℃, the flow rate was ±0.2mL/min, the pH of the mobile phase was ±0.1, and different chromatographic columns were verified. Take the system suitability solution, the impurity reference solution and the mixed sample solution respectively for testing under various conditions, and perform statistical analysis on the impurity content. The test results are shown in Table 6 and Table 7.

Table 6 Impurity 1 durability test results

Table 7 Impurity 2 Durability Test Results

According to the test results of the above examples, the method for detecting potential mutagenic impurities of pitavastatin calcium tablets of the present invention is simple to operate, easy to control, high in sensitivity, and has good specificity, system applicability, solution stability and chromatography. Conditional durability provides an effective detection method for monitoring product quality and further guarantees product safety.

Claims (10)

1. A method for detecting potential mutagenic impurities in pitavastatin calcium tablets, which is characterized in that: after the sample to be tested is dissolved in a solvent, high performance liquid chromatography is used for detection, and the chromatographic conditions are as follows:

   Mobile phase: organic acid salt buffer and acetonitrile;

   Flow rate: 1.0mL/min;

   Column temperature: 25～40℃;

   Chromatographic column: use octadecyl silane bonded silica gel as filler;

   Injection volume: 50μL;

   Detection wavelength: 270～280nm;

   Elution method: gradient elution;

   Among them, the potential mutagenic impurities are impurity 1 and impurity 2, and the structure of impurity 1 is:

   

   The structure of impurity 2 is:

   
2. The method for detecting potentially mutagenic impurities in pitavastatin calcium tablets according to claim 1, characterized in that: the detection is carried out by high performance liquid chromatography, a reversed-phase high performance liquid chromatography system, and a gradient elution procedure are used. as follows:

   
3. The method for detecting potential mutagenic impurities in pitavastatin calcium tablets according to claim 1, wherein the organic acid salt buffer is an acetate buffer with a pH of 3.4-3.6.
4. The method for detecting potential mutagenic impurities in pitavastatin calcium tablets according to claim 1, wherein the solvent is acetonitrile-water solution, and the volume content of acetonitrile is 40-60%.
5. The method for detecting potentially mutagenic impurities in pitavastatin calcium tablets according to claim 1, wherein the column temperature is 30°C.
6. The method for detecting potentially mutagenic impurities in pitavastatin calcium tablets according to claim 1, wherein the detection wavelength is 275 nm.
7. The method for detecting potentially mutagenic impurities in pitavastatin calcium tablets according to claim 1, characterized in that it comprises the following steps:

   (1) Preparation of sample solution;

   (2) Preparation of system suitability solution;

   (3) Preparation of impurity reference substance solution;

   (4) Detection method: adopt high performance liquid chromatography, use gradient elution procedure for impurity separation, and control impurity content by limit method.
8. The method for detecting potential mutagenic impurities in pitavastatin calcium tablets according to claim 7, characterized in that: in step (1), the preparation method of the sample solution is: taking a sample equivalent to 20 mg of pitavastatin calcium, Add solvent, dissolve by ultrasonic, dilute to a constant volume of 40 mL, centrifuge or stand still, and take the supernatant to obtain the sample solution.
9. The method for detecting potential mutagenic impurities in pitavastatin calcium tablets according to claim 7, characterized in that: in step (2), the preparation method of the system suitability solution is: weighing pitavastatin calcium reference substance, Vastatin calcium impurity 1 reference substance, pitavastatin calcium impurity 2 reference substance, dissolved and diluted with a solvent to prepare a mixed solution containing pitavastatin calcium 0.5mg/mL, impurity 10.15μg/mL, impurity 2 0.15μg/mL , The system suitability solution is obtained.
10. The method for detecting potential mutagenic impurities in pitavastatin calcium tablets according to claim 7, characterized in that: in step (3), the preparation method of the impurity reference substance solution is: weighing pitavastatin calcium impurity 1 reference substance , Impurity 2 reference substance, dissolve and dilute with a solvent to prepare a mixed solution containing 1 0.15μg/mL of impurities and 2 0.15μg/mL of impurities to obtain the solution of impurity reference substance.

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