CN110568121A

CN110568121A - Eribulin and detection method of related substances in eribulin-containing preparation

Info

Publication number: CN110568121A
Application number: CN201910626874.0A
Authority: CN
Inventors: 付丙月; 张雯; 王金虎; 张宁; 孙玲; 孙青�; 王颖超; 吴伟山
Original assignee: Shandong Academy of Pharmaceutical Sciences
Current assignee: Shandong Academy of Pharmaceutical Sciences
Priority date: 2019-07-11
Filing date: 2019-07-11
Publication date: 2019-12-13

Abstract

The invention discloses eribulin and a detection method of related substances in a preparation containing eribulin. The method specifically adopts reversed-phase high performance liquid chromatography, rapidly and accurately detects the impurity and degradation product conditions of eribulin, solves the problem that various impurities generated in the synthesis and preparation processes interfere with the product purity, and can comprehensively and reliably control the quality of the raw material medicine and the preparation. The method has the advantages of simple operation, high sensitivity and good separation degree, and provides an effective analysis method for controlling the product quality.

Description

eribulin and detection method of related substances in eribulin-containing preparation

Technical Field

The invention relates to eribulin and a detection method of related substances in a preparation containing eribulin, and belongs to the field of drug analysis and detection.

Background

Eribulin (chemical name: Eribulin, trade name: Halaven), having the following chemical formula:

11 months 2010, approved by the U.S. FDA for the treatment of metastatic breast cancer, was the first approved drug for liposarcoma and demonstrated improvement in survival. Eribulin has an anticancer action mechanism similar to that of paclitaxel, and can block the G2/M cell circuit through a tubulin antimitotic pathway, affect mitotic spindle, and have no influence on microtubule shortening and microtubule division into non-secretory aggregates, and finally, the mitotic process is blocked, resulting in cell death. It is now used clinically for the treatment of liposarcoma (a particular form of soft tissue sarcoma) that cannot be removed by surgery (unresectable) or at an advanced stage (metastatic).

At present, the medicine does not have a unified detection standard in China, so in order to better control the quality of eribulin products, a set of simple, reliable, stable and effective method needs to be established to detect related substances of the eribulin products, and the problem of interference of various known or unknown impurities introduced by a synthesis process or generated in degradation and preparation processing processes on product purity determination is solved.

The invention adopts an isocratic elution mode, effectively detects the content and related substances of eribulin and preparations containing eribulin, can quickly and accurately detect impurities and degradation products of eribulin, has simple and convenient operation and high sensitivity, and can better control the product quality. The invention realizes the measurement of eribulin and related substances of preparations containing eribulin, ensures the quality control of eribulin, and has important practical significance in the aspects of synthesis and quality control of preparation processes.

disclosure of Invention

the invention aims to establish a method for detecting related substances in eribulin and preparations containing eribulin. The method overcomes the problem that the medicine has no unified medicine standard, solves the interference of various known or unknown impurities introduced by a synthesis process or generated in degradation and preparation processing processes on product purity determination, and better controls the quality of eribulin products. Considering that normal phase chromatography is not suitable for a liquid chromatograph-mass spectrometer, the separation and structure qualitative detection of eribulin and related substances of preparations containing eribulin cannot be further carried out, and therefore reverse phase high performance liquid chromatography is researched. The method has the advantages that: 1. the related substances can be effectively separated, and quantitative control can be realized; 2. the method has universal practicability; 3. sufficient sensitivity to effectively control impurities.

The invention provides eribulin and a method for detecting related substances in preparations containing eribulin, which comprises the following steps: (1) dissolving eribulin raw material medicine or preparation sample containing eribulin with mobile phase to prepare test solution; (2) adopting reversed-phase high performance liquid chromatography, wherein the chromatographic column is a C18 column, the column temperature is 15-40 ℃, the flow rate is 0.5-1.5 ml/min, polar organic solvent-inorganic salt buffer solution is used as a mobile phase, and isocratic elution is adopted; (3) and (3) detecting the wavelength of 230-260 nm by adopting an ultraviolet detector, and recording the chromatogram to finish the detection of the eribulin and the related substances in the preparation containing the eribulin.

Furthermore, in the detection method, eribulin and a preparation sample containing eribulin are dissolved and diluted by using a mobile phase to prepare a test solution containing 0.01-5 mg of eribulin per 1 ml.

Wherein, the mixed solution of acetonitrile-acetate buffer solution or the mixed solution of methanol-acetate buffer solution is used as a mobile phase, and the volume ratio of the acetonitrile-acetate buffer solution to the methanol-acetate buffer solution is 95%: 5% -80%: 20 percent.

Furthermore, the concentration of the acetate buffer solution is 0.001-0.1 mol/L, the pH value is 3.5-7.0, wherein the concentration is preferably 0.001-0.05 mol/L, the pH value is 5.0-6.5, the optimal concentration is 0.01mol/L, and the optimal pH value is 5.0 or 6.0. The salt types of the acetate buffer include: ammonium acetate, sodium acetate, potassium acetate, calcium acetate, zinc acetate, magnesium acetate, and the like, among which ammonium acetate, sodium acetate, and potassium acetate are preferable through experiments, and an ammonium acetate buffer solution is most preferable.

In the detection method, a chromatographic column is Eclipse XDB-C18.

In the detection method, the temperature of the chromatographic column is 15-40 ℃.

In the detection method of the invention, the detection wavelength is 254 nm.

Compared with the prior art, the invention has the positive effects that:

1. the liquid chromatography provided by the invention has stronger superiority, is suitable for detection of crude drugs and related substances of various dosage forms thereof, and can quickly and accurately detect the impurity and degradation product conditions of eribulin. In the detection of related substances of eribulin raw materials and preparations thereof, the peak shapes are symmetrical, the separation degree is high, and the problems of the appearance of a front peak, insufficient separation degree of individual peaks and the like are avoided.

2. The invention uses the ultraviolet detector to detect related substances of the product, and establishes effective prevention for rapidly and visually evaluating the purity of the chromatographic peak. The method can visually judge the purity of the chromatographic peak and judge the position with interference fee, can obtain all spectrum information of 'flow' and quickly obtain the absorption spectrum of chromatographic components, and has the advantages of not only depending on the retention time of the chromatogram for qualitative determination, but also being capable of performing qualitative determination according to the spectrum information provided by the chromatogram, thereby greatly improving the qualitative reliability and the detection sensitivity.

3. the detection method of the invention has the advantages of accuracy, simple operation, good reproducibility and high sensitivity, can fully meet the requirements of related substance detection and decomposition product determination, well control the special impurities in the sample, ensure the product quality and have strong practicability in work. The method is beneficial to the detection of relevant substances in eribulin and preparations containing eribulin in industrial production, and can better control the product quality and ensure the safety of medicines.

Drawings

FIG. 1 is a linear relationship diagram of the impurity limit of eribulin

FIG. 2-1. liquid chromatogram of eribulin control

FIG. 2-2 is a liquid chromatogram of related substances in eribulin raw material

FIG. 3 is a liquid chromatogram of related substances of eribulin dimesylate injection

Detailed Description

The invention will now be further described by way of the following examples, which are not intended to limit the scope of the invention.

Example an Eribulin-related substance assay

1. Chromatographic conditions and System suitability test

1.1 selection of chromatographic conditions:

The instrument comprises the following steps: agilent 1260, with an optimal column temperature of 35 deg.C, flow rate of 1.0ml/min, and detection wavelength of 254 nm. Liquid chromatography column Eclipse XDB-C18 column (150 mm. times.4.6 mm, 5 μm), 0.01mol/L NH₄OAc solution (pH5.0) -acetonitrile or 0.01mol/L NH₄OAc solution (pH6.0) -methanol is used as a mobile phase composition, and the optimal mixture ratio is as follows: 10:90 (mobile phase composition order is consistent with volume ratio order). The amount of the sample was 20. mu.l.

Under the chromatographic condition, the retention time of the main peak of eribulin is moderate, and the peak shape is better.

1.2 sensitivity determination

Taking a proper amount of eribulin, preparing a solution with the concentration of 0.2mg in each 1ml by using a mobile phase, diluting the solution into a series of solutions with different concentrations by using the mobile phase respectively, and injecting 10 mu l of the solution respectively to generate a signal with a main peak three times the baseline noise. The test shows that the minimum detection amount is 0.2ng (S/N is more than or equal to 3), and the detection limit is 0.1% when the concentration of the relevant substances in the test is 0.2 mg/ml. The result proves that the method has high sensitivity and can fully meet the requirements of checking and measuring related substances.

1.3 solution stability

The same sample solution is taken and respectively injected for measurement in 0 hour, 2 hours, 4 hours, 8 hours, 10 hours and 12 hours, and the peak area of the main peak and the measurement result of related substances are basically stable within 12 hours.

the experimental results show that the method is simple, convenient and sensitive, has good reproducibility, and can be used for better detecting the quality of eribulin in the sample.

1.4 preparing a test solution;

Taking a proper amount of eribulin raw material, and adding a mobile phase to prepare a solution of 0.2mg/ml, wherein the solution is used as a test solution.

1.5 preparation of control solutions:

An appropriate amount of the above 1.4 sample solution was measured and diluted with a mobile phase to a solution containing about 2. mu.g of the sample solution per 1ml, which was used as a control solution. The control solution concentration ranges were: when the concentration is within 1-20 mu g/ml, the peak area and the concentration form a good linear relation, and the linear correlation coefficient is 0.9998. See attached figure 1

2. Detecting related substances of eribulin raw materials:

And (2) taking 10ul of the test solution, injecting the test solution into a liquid chromatograph, adjusting the detection sensitivity to enable the main component chromatographic peak height to be about 10% of the full range, then taking 10ul of the test solution, injecting the test solution into the liquid chromatograph, recording a chromatogram, and measuring the sum of the peak areas of the impurities if the impurity peaks exist in the chromatogram of the test solution, wherein the sum of the peak areas of the impurities is not larger than the peak area of the main peak in the control solution. See figure 2

Example two: determination of related substances of eribulin mesylate injection

Taking a test sample, precisely measuring a proper amount (about 2mg of eribulin), adding a mobile phase to prepare a solution containing 0.2mg of the mobile phase in each 1ml, filtering, and taking a filtrate of a diluent as a test sample solution. An appropriate amount was precisely measured, and diluted with a mobile phase to a solution containing about 2. mu.g per 1ml, as a control solution. Under chromatographic conditions selected as follows:

An ultraviolet detector (Agilent 1260) for detecting 254nm wavelength, Eclipse XDB-C18 column (150mm × 4.6mm, 5 μm), 0.01mol/L NH₄OAc solution (pH6.0) -acetonitrile, the optimal mixture ratio is: 10:90. The column temperature of the chromatographic column is 30 ℃, and the flow rate is 1.0 ml/min. The amount of the sample was 20. mu.l. Calculated according to eribulin peak, on theoretical plate, is not less than 3000.

the results of the measurements are shown in the following table:

Batch number	first batch	Second batch	Third batch
				Related substance (%)	0.11	0.15	0.16

Claims

1. A method for detecting eribulin and related substances in preparations containing eribulin is characterized by comprising the following steps:

(1) Dissolving a eribulin raw material medicine or a preparation sample containing eribulin by using a mobile phase to prepare a test solution, and preparing a test solution containing 0.01-5 mg of eribulin per 1 ml;

(2) Adopting reversed-phase high performance liquid chromatography, wherein a chromatographic column is a C18 column, the flow rate is 0.5-1.5 ml/min, a mixed solution of a polar organic solvent and an inorganic salt buffer solution is used as a mobile phase, and isocratic elution is adopted;

(3) And (3) detecting the wavelength of 230-260 nm by adopting an ultraviolet detector, and recording the chromatogram to finish the detection of the eribulin and the related substances in the preparation containing the eribulin.

2. The detection method according to claim 1, wherein in the step (2), the temperature of the chromatographic column is 15 to 40 ℃.

3. The detection method according to claim 1, wherein in the step (2), the chromatography column is selected from Eclipse XDB-C18.

4. The assay of claim 1, wherein in step (2), the buffered salt solution is selected from an acetate buffer.

5. The detection method according to claim 1, wherein in the step (2), the pH value of the acetate buffer solution is 3.5 to 7.0.

6. The detection method according to claim 1, wherein the volume ratio of the polar organic solvent-acetate buffer solution in the mobile phase in the step (2) is 95%: 5% -80%: 20 percent.

7. The detection method according to claim 1, wherein the polar organic solvent in the mobile phase in the step (2) is selected from acetonitrile or methanol.

8. The detection method according to claim 5, wherein the concentration of the acetate buffer is 0.001 to 0.1 mol/L.

9. The detection method according to claim 1, wherein in the step (2), the flow rate is 1.0 ml/min.

10. The detection method according to claim 1, wherein in the step (3), the detection wavelength is 254 nm.