CN110568121A - A method for detecting related substances in eribulin and eribulin-containing preparations - Google Patents

Abstract

The invention discloses a method for detecting related substances in eribulin and preparations containing eribulin. This method specifically uses reversed-phase high-performance liquid chromatography to quickly and accurately detect the impurities and degradation products of eribulin, and solves the interference of various impurities generated in the process of its synthesis and preparation on the purity of the product, which can be comprehensive and reliable. To accurately control the quality of raw materials and preparations. The method is simple to operate, high in sensitivity and good in resolution, and provides an effective analytical method for controlling product quality.

Description

A method for detecting related substances in eribulin and eribulin-containing preparations

technical field

The invention relates to a method for detecting related substances in eribulin and preparations containing eribulin, belonging to the field of drug analysis and detection.

Background technique

Eribulin (chemical name: Eribulin, trade name: Halaven), the chemical structural formula is as follows:

In November 2010, it was approved by the US FDA for the treatment of metastatic breast cancer. It was the first drug approved for liposarcoma and proved to improve survival. Eribulin’s anti-cancer mechanism is similar to that of paclitaxel. It can block the G2/M cell circuit through the anti-mitotic pathway of tubulin and affect the mitotic spindle without affecting the shortening period of microtubules and the division of tubulin into non- secreted aggregates, and finally the mitotic process is blocked, leading to cell death. It is now used clinically for the treatment of liposarcoma (a specific form of soft tissue sarcoma) that cannot be removed by surgery (unresectable) or advanced (metastatic).

At present, there is no unified detection standard for this drug in China. Therefore, in order to better control the quality of Eribulin products, it is necessary to establish a set of simple, reliable, stable and effective methods to detect the related substances of this product and solve the problem of synthetic process. The interference of various known or unknown impurities introduced or produced during degradation and preparation processing on the determination of product purity.

The invention adopts an isocratic elution method to effectively detect the content and related substances of eribulin and preparations containing eribulin, and can quickly and accurately detect impurities and degradation products of eribulin, and the operation is simple and convenient , High sensitivity, can better control product quality. The invention realizes the determination of related substances of eribulin and preparations containing eribulin, ensures the quality control of eribulin, and has important practical significance in the aspects of synthesis and quality control of the preparation process.

Contents of the invention

The purpose of the present invention is to establish a detection method capable of controlling related substances in eribulin and eribulin-containing preparations. This method overcomes the problem that the drug does not have a uniform drug standard, solves the interference of various known or unknown impurities introduced by the synthesis process or generated by degradation and preparation processing on the determination of product purity, and better controls Eribulin products. the quality of. Considering that the adjustment of normal-phase chromatography is not suitable for liquid chromatography-mass spectrometry, the separation and structural qualitative detection of related substances in eribulin and eribulin-containing preparations cannot be further carried out, so reversed-phase high-performance liquid chromatography was studied. The advantages of this method are: 1. It can effectively separate related substances and can quantitatively control them; 2. This method has universal practicability; 3. It has sufficient sensitivity and can effectively control impurities.

The invention provides a method for detecting related substances in eribulin and eribulin-containing preparations, the steps are as follows: (1) take eribulin bulk drug or eribulin-containing preparation samples, and use mobile phase Dissolve and prepare the test solution; (2) adopt reversed-phase high-performance liquid chromatography, the chromatographic column is a C18 column, the column temperature is 15°C to 40°C, and the flow rate is 0.5 to 1.5ml/min, with a polar organic solvent-inorganic salt The buffer solution is the mobile phase, and isocratic elution is adopted; (3) an ultraviolet detector is used, and the detection wavelength is 230-260 nm, and the chromatogram is recorded to complete the detection of related substances in eribulin and eribulin-containing preparations.

Further, in the detection method of the present invention, the samples of eribulin and preparations containing eribulin are dissolved and diluted with 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 or the mixed solution of methanol-acetate buffer is used as the mobile phase, and the volume ratio of the two is 95%:5%-80%:20%.

Furthermore, the concentration of 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, and the optimal concentration is 0.01 mol/L L, the optimum pH 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, etc. Among them, ammonium acetate, sodium acetate, and potassium acetate are preferred through experiments, and ammonium acetate buffer is most preferred.

In the detection method of the present invention, the chromatographic column is Eclipse XDB-C18.

In the detection method of the present invention, the temperature of the chromatographic column is between 15°C and 40°C.

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

The positive effect of the present invention compared with prior art is:

1. The superiority of liquid chromatography conditions in the present invention is stronger, and it is suitable for the detection of related substances of raw materials and various dosage forms thereof, and can quickly and accurately detect impurities and degradation products of eribulin. In the detection of related substances of eribulin raw materials and its preparations, the peak shapes are symmetrical and the resolution is high, which avoids problems such as frontier peaks and insufficient resolution of individual peaks.

2. The present invention uses an ultraviolet detector to detect related substances in the product, and establishes an effective prevention method for quickly and intuitively evaluating the purity of chromatographic peaks. It can visually judge the purity of chromatographic peaks and judge the position of interference charges. It can obtain all the spectral information of "in-flow" and quickly obtain the absorption spectrum of chromatographic components. It can perform qualitative according to the spectral information provided by it, which greatly improves the reliability of the qualitative and the detection sensitivity.

3. The detection method of the present invention is accurate, easy to operate, good in reproducibility and high in sensitivity, can fully meet the requirements of the detection of related substances and the determination of decomposition products, relatively well control the special impurities in the sample, ensure the quality of the product, and be practical in work Strong. It is beneficial to the detection of related substances in eribulin and preparations containing eribulin in industrialized production, and can better control product quality and ensure drug safety.

Description of drawings

Figure 1. Eribulin impurity limit linear relationship diagram

Figure 2-1. The liquid chromatogram of the Eribulin reference substance in Example 1

Figure 2-2. Liquid chromatogram of related substances of eribulin raw material

Figure 3. Liquid chromatogram of related substances in Eribulin dimesylate injection of the embodiment

Detailed ways

The present invention will be further described by the following examples, but these descriptions are not further limitations to the content of the present invention.

Embodiment 1 Determination of Eribulin Related Substances

1. Chromatographic conditions and system suitability experiments

1.1 Selection of chromatographic conditions:

Instrument: Agilent 1260, the optimum column temperature is 35°C, the flow rate is 1.0ml/min, and the detection wavelength is 254nm. Eclipse XDB-C18 column (150mm×4.6mm, 5μm) for liquid chromatography, 0.01mol/L NH ₄ OAc solution (pH5.0)-acetonitrile or 0.01mol/L NH ₄ OAc solution (pH6.0)-methanol It is the composition of the mobile phase, and its optimal ratio is: 10:90 (the sequence of the composition of the mobile phase is consistent with the sequence of the volume ratio). The injection volume is 20 μl.

Under this chromatographic condition, the main peak of eribulin had a moderate retention time and a good peak shape.

1.2 Sensitivity determination

Take an appropriate amount of eribulin, use the mobile phase to prepare a solution with a concentration of 0.2 mg per 1 ml, and then use the mobile phase to dilute it into a series of solutions with different concentrations, and inject 10 μl of the sample respectively, so that the main peak is three times the noise of the baseline. Signal. According to the test, the minimum detection amount is 0.2ng (S/N≥3), if calculated based on the concentration of 0.2mg/ml when the related substances are checked, the detection limit is 0.1%. The results show that the method has high sensitivity and can fully meet the requirements of the inspection and determination of related substances.

1.3 Solution stability

Take the same part of the test product solution and inject samples respectively at 0, 2, 4, 8, 10, and 12 hours for determination, and the peak area of the main peak and the determination results of related substances are basically stable within 12 hours.

The above experimental results show that this method is simple, sensitive, and reproducible, and can better detect the quality of eribulin in the sample.

1.4 Configuration of the test solution;

Take an appropriate amount of eribulin raw material, add mobile phase to configure a 0.2mg/ml solution, as the test solution.

1.5 Preparation of reference solution:

Take an appropriate amount of the above-mentioned 1.4 test solution, add mobile phase and dilute it to a solution containing about 2 μg per 1 ml, as a control solution. When the concentration range of the control solution is in the range of 1-20 μg/ml, the peak area has a good linear relationship with the concentration, and the linear correlation coefficient is 0.9998. See Attachment 1

2. Detection of related substances in Eribulin raw materials:

Get need testing solution 10ul, inject liquid chromatograph, adjust detection sensitivity, make the chromatographic peak height of main component be about 10% of full scale, then get need testing solution 10ul inject liquid chromatograph, record chromatogram, for testing If there are impurity peaks in the product solution chromatogram, measure the sum of the peak areas of each impurity, which shall not be greater than the peak area of the main peak in the reference solution. See Attachment 2

Embodiment Two: Determination of Related Substances in Eribulin Mesylate Injection

Get the test product, accurately measure an appropriate amount (approximately equivalent to eribulin 2 mg), add mobile phase to configure a solution containing 0.2 mg per 1 ml, filter, and take the diluted filtrate as the test product solution. Precisely measure an appropriate amount, add mobile phase to dilute to a solution containing about 2 μg per 1 ml, and use it as a control solution. Under the following selected chromatographic conditions:

Ultraviolet detector (Agilent 1260) detection wavelength 254nm, Eclipse XDB-C18 column (150mm×4.6mm, 5 μm), 0.01mol/L NH ₄ OAc solution (pH6.0)-acetonitrile composition, the optimal ratio is: 10:90. The column temperature of the chromatographic column is 30°C, and the flow rate is 1.0ml/min. The injection volume is 20 μl. The theoretical plate is not less than 3000 based on Eribulin peak.

The measurement results are shown in the table below:

batch number first batch second batch third batch relative substance(%) 0.11 0.15 0.16

Claims (10)

1. A detection method for related substances in eribulin and preparations containing eribulin, characterized in that the detection method comprises the following steps: (1) Take the eribulin bulk drug or the preparation sample containing eribulin, dissolve it in mobile phase, prepare the test solution, and make a test solution containing 0.01-5 mg eribulin per 1 ml; (2) Reversed-phase high-performance liquid chromatography, the chromatographic column is a C18 column, the flow rate is 0.5-1.5ml/min, and the mixed solution of polar organic solvent-inorganic salt buffer is used as the mobile phase, and isocratic elution is adopted; (3) Use an ultraviolet detector with a detection wavelength of 230-260 nm, record the chromatogram, and complete the detection of related substances in the eribulin and the preparation containing eribulin. 2. The detection method according to claim 1, characterized in that, in the step (2), the temperature of the chromatographic column is between 15°C and 40°C. 3. detection method according to claim 1, is characterized in that, in described step (2), chromatographic column is selected from EclipseXDB-C18. 4. The detection method according to claim 1, characterized in that, in the step (2), the buffered saline solution is selected from acetate buffer. 5. The detection method according to claim 1, characterized in that, in the step (2), the pH value of the acetate buffer is 3.5-7.0. 6. detection method according to claim 1, is characterized in that, the volume ratio of polar organic solvent-acetate buffer solution in mobile phase in described step (2) is 95%: 5%～80%: 20 %. 7. detection method according to claim 1, is characterized in that, in described step (2), polar organic solvent in mobile phase is selected from acetonitrile or methanol. 8. The detection method according to claim 5, characterized in that the concentration of the acetate buffer is 0.001-0.1 mol/L. 9. The detection method according to claim 1, characterized in that, in the step (2), the flow rate is 1.0ml/min. 10. The detection method according to claim 1, characterized in that, in the step (3), the detection wavelength is 254nm.