阅读说明：本技术 一种曲伏前列素有关物质的检测方法 (Detection method of travoprost related substances ) 是由 陈立营 张成飞 方路平 王淑丽 王大庆 于 2021-01-15 设计创作，主要内容包括：本发明提供了一种曲伏前列素有关物质的检测方法,采用高效液相色谱法,色谱条件包括：采用十八烷基硅烷键合硅胶填充剂的色谱柱,紫外检测器检测,流动相由流动相A和流动相B组成,所述流动相A为磷酸水溶液,流动相B为乙腈-甲醇,梯度洗脱。本发明通过对流动相、洗脱梯度、流速及柱温的优化设计,使曲伏前列素、15-epi非对映异构体、5,6反式异构体达到基线分离,可有效测定15-epi非对映异构体、5,6反式异构体及其他有关物质含量,确保曲伏前列素及其制剂的质量可控。另外,此方法中所用流速相对较小且流动相不含缓冲盐,不易堵塞色谱柱,可提高色谱柱使用寿命和仪器耐用性。(The invention provides a method for detecting travoprost related substances, which adopts high performance liquid chromatography, and the chromatographic conditions comprise: detecting by using a chromatographic column of octadecylsilane bonded silica gel filler and an ultraviolet detector, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is acetonitrile-methanol, and gradient elution is carried out. By optimally designing a mobile phase, an elution gradient, a flow rate and a column temperature, the invention enables the travoprost, the 15-epi diastereoisomer and the 5,6 trans isomer to achieve baseline separation, can effectively measure the contents of the 15-epi diastereoisomer, the 5,6 trans isomer and other related substances, and ensures the controllable quality of the travoprost and the preparation thereof. In addition, the flow rate used in the method is relatively small, and the mobile phase does not contain buffer salt, so that the chromatographic column is not easy to block, and the service life of the chromatographic column and the durability of the instrument can be improved.)

1. The detection method of the travoprost related substances is characterized in that high performance liquid chromatography is adopted, and the chromatographic conditions comprise: detecting by using a chromatographic column of octadecylsilane bonded silica gel filler and an ultraviolet detector, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is acetonitrile-methanol, and gradient elution is carried out.

The gradient is set as follows:

2. adopting high performance liquid chromatography, wherein the chromatographic conditions comprise: the method comprises the following steps of detecting by using a chromatographic column of octadecylsilane bonded silica gel filler and an ultraviolet detector, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is acetonitrile-methanol, and the gradient is set as follows:

3. the method for detecting a travoprost-related substance according to claim 1 or 2, wherein the volume ratio of acetonitrile to methanol in the mobile phase B is (95-98): (2-5).

4. The method for detecting a travoprost-related substance according to claim 3, wherein the volume ratio of acetonitrile to methanol in the mobile phase B is 98: 2.

5. the method for detecting a travoprost-related substance according to any one of claims 1, 2, or 4, wherein the concentration of phosphoric acid in mobile phase A is 0.04% to 0.06%.

6. The method for detecting a travoprost-related substance according to claim 5, wherein the concentration of phosphoric acid in mobile phase A is 0.05%.

7. The method as claimed in claim 1, 2, 4 or 6, wherein the detection wavelength used is 222 nm.

8. The method for detecting a travoprost-related substance according to claim 7, wherein the column temperature is set to 28 ℃ to 32 ℃.

9. The method for detecting a travoprost-related substance according to any one of claims 1, 2, 4, 6, or 8, wherein the flow rate is set to 2.0mL/min to 3.0 mL/min.

10. The method for detecting a travoprost-related substance according to claim 9, wherein the flow rate is set to 2.0mL/min to 2.5 mL/min.

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a detection method of a kovoprost related substance.

Background

Travoprost is an isopropyl ester prodrug that is hydrolyzed by corneal hydrolases to the active free acid after ocular administration, distributed in ocular tissues, and binds to activate the PG-FP receptors located in ciliary muscle and trabecular cells. This activation can trigger a cascade of intracellular signaling events leading to the production of inositol triphosphates and diglycerides and mobilizing intracellular calcium ions which in turn increases the release of Matrix Metalloproteinase (MMP) precursors in the cell. MMP can degrade extracellular matrix in the aqueous outflow path of uveal sclera, reduce outflow resistance, promote aqueous outflow through the channel of uveal sclera, reduce intraocular pressure, and the preparation is travoprost eye drops.

The travoprost related substances are more, and mainly comprise a travoprost related compound A, an epoxidized derivative, a 15-epi diastereoisomer, a 5, 6-trans isomer and a 15-ketone derivative. Wherein, the 15-epi diastereoisomer and the 5,6 trans isomer are isomers of travoprost, and the polarity of the 15-epi diastereoisomer and the 5,6 trans isomer is similar and not easy to separate, which increases the difficulty of quality control of the travoprost.

The detection method of related substances recorded in the USP42 pharmacopoeia of travoprost has a plurality of problems, for example, the separation degree between chromatographic peaks of 15-epi diastereoisomer and travoprost is less than 1.5 by adopting the method of the United states pharmacopoeia, and the retention time of chromatographic columns in different batches is different; the adoption of buffer salt mobile phase and high flow rate, easy consumption of chromatographic column and reduced column efficiency.

In the related substance detection method loaded in the USP42 pharmacopoeia of travoprost eye drops, different impurities are detected by adopting different mobile phases, the detection is at least twice, and the method is complicated.

The related substance analysis method carried by the imported registration standard (JX20170195) of the travoprost eye drops adopts ultra-high efficiency, the concentration of a test sample is not suitable to be overlarge, the concentration is slightly high, and an overload phenomenon occurs, so that the separation degree between a main peak and an adjacent impurity peak is not in accordance with the regulation, and the quality of the test sample cannot be accurately and effectively controlled.

In addition, there is a literature reporting on the analysis method of travoprost, but there is no indication on the research situation of adjacent impurities of travoprost, and the research systems such as wavelength, mobile phase and pharmacopoeia system are greatly different.

Therefore, a new analysis method needs to be established, so that the running time can be shortened, and the service life of the chromatographic column can be prolonged; and various related substances can be simultaneously separated and detected, particularly, effective separation among 15-epi diastereoisomer, travoprost and 5,6 trans isomer is realized, and the method has important significance on quality control of travoprost raw materials and preparations thereof.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The main object of the present invention is to provide a method for detecting travoprost-related substances, which is intended to at least partially solve at least one of the above-mentioned technical problems.

The related substances comprise a travoprost related compound A, an epoxidized derivative, a 15-epi diastereoisomer, a 5, 6-trans isomer and a 15-ketone derivative, and the specific structural formula is as follows:

the invention provides a method for detecting travoprost related substances, which adopts high performance liquid chromatography, and the chromatographic conditions comprise: detecting by using a chromatographic column of octadecylsilane bonded silica gel filler and an ultraviolet detector, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is acetonitrile-methanol, and gradient elution is carried out.

The gradient is set as follows:

further, the chromatographic conditions include: the method comprises the following steps of detecting by using a chromatographic column of octadecylsilane bonded silica gel filler and an ultraviolet detector, wherein a mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is acetonitrile-methanol, and the gradient is set as follows:

further, the volume ratio of acetonitrile to methanol in the mobile phase B is (95-98): (2-5).

Further, the volume ratio of acetonitrile to methanol in the mobile phase B is 98: 2.

further, the concentration of phosphoric acid in the mobile phase A is 0.04-0.06%.

Further, the concentration of phosphoric acid in the mobile phase a was 0.05%.

Further, the detection wavelength used was 218-222 nm.

Further, the detection wavelength used was 220 nm.

Further, the column temperature was set to 28 ℃ to 32 ℃.

Further, the column temperature was set to 30 ℃.

Further, the flow rate used was set to 2.0mL/min to 3.0 mL/min.

Further, the flow rate used was set to 2.0mL/min to 2.5 mL/min. Further, the phosphoric acid is chromatographic grade phosphoric acid.

The baseline noise of the chromatogram was different for different grades of phosphoric acid. The improper selection of the phosphoric acid causes the base line of the chromatogram to have larger noise and uneven base line, and has interference on impurity detection.

Compared with the prior art, the invention has the following beneficial effects:

according to the method for detecting the travoprost related substances, provided by the invention, by optimally designing the mobile phase, the elution gradient, the flow rate and the column temperature, the travoprost, the 15-epi diastereoisomer and the 5,6 trans isomer can be subjected to baseline separation, the contents of the 15-epi diastereoisomer, the 5,6 trans isomer and other related substances can be effectively measured, and the quality of the travoprost and the preparation thereof can be controlled. In addition, the flow velocity is relatively small, the mobile phase does not contain buffer salt, the chromatographic column is not easy to block, and the service life of the chromatographic column and the durability of the instrument can be improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an HPLC chromatogram of the mixed control solution of example 1;

FIG. 2 is an HPLC chromatogram of a localization solution of travoprost-related Compound A in example 1;

FIG. 3 is an HPLC chromatogram of a localization solution of the epoxidized derivative in example 1;

FIG. 4 is an HPLC chromatogram of a 15-epi diastereomer localization solution of example 1;

FIG. 5 is an HPLC chromatogram of a 5, 6-trans isomer positioning solution of example 1;

FIG. 6 is an HPLC chromatogram of a 15-ketone derivative localization solution in example 1;

FIG. 7 is an HPLC chromatogram of a travoprost localization solution of example 1;

FIG. 8 is the durability HPLC spectrum of example 2 at 30 ℃ C;

FIG. 9 is the durability HPLC spectrum of example 2-column temperature 28 ℃;

FIG. 10 is the durability HPLC spectrum of example 2 at a column temperature of 32 ℃;

FIG. 11 is a graph of the durability HPLC spectrum of example 2 at wavelength 218 nm;

FIG. 12 is a graph of the durability HPLC spectrum of example 2 at a wavelength of 222 nm;

FIG. 13 is an HPLC chromatogram of the detection limit of 5, 6-trans isomer in example 3;

FIG. 14 is an HPLC chromatogram of the detection limit of travoprost in example 3;

FIG. 15 is an HPLC chromatogram of the limit of detection of travoprost-related Compound A in example 3;

FIG. 16 is an HPLC chromatogram showing the detection limit of epoxidized derivatives in example 3;

FIG. 17 is an HPLC chromatogram of the detection limit of the 15-epi diastereomer in example 3;

FIG. 18 is an HPLC chromatogram showing the detection limit of the 15-ketone derivative in example 3.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples were carried out under the conventional conditions, unless otherwise specified. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Travoprost-related compound a, epoxidized derivatives, 15-epi diastereomer, 5, 6-trans isomer, and 15-ketone derivative controls were purchased ex vivo.

Example 1 specificity test

The formulation of the solution is detailed in table 1.

TABLE 1 preparation of the solutions

Chromatographic conditions are as follows:

a chromatographic column: ODSHYPERSIL (4.6 mm. times.50 mm,3 μm);

mobile phase A: 0.05% aqueous phosphoric acid;

mobile phase B: acetonitrile-methanol (98: 2);

detection wavelength: 220 nm;

flow rate: 2.5 mL/min;

column temperature: 30 ℃;

sample introduction amount: 5 mu L of the solution;

gradient elution was performed according to the following table:

the detection data are shown in a table 2, and the atlas is shown in figures 1-7.

TABLE 2 results for mixed control and positioning solutions

As can be seen from Table 2, the degree of separation between each known impurity and the adjacent impurity is greater than 1.5, which meets the requirements.

EXAMPLE 2 durability test

The formulation of the solution is detailed in table 3.

TABLE 3 preparation of the solutions

Chromatographic conditions are as follows:

a chromatographic column: ODSHYPERSIL (4.6 mm. times.50 mm,3 μm);

mobile phase A: 0.05% aqueous phosphoric acid;

mobile phase B: acetonitrile-methanol (98: 2);

flow rate: 2.0 mL/min;

sample introduction amount: 5 mu L of the solution;

gradient elution was performed according to the following table:

the detection data are shown in Table 4, and the map is shown in figures 8-12.

Table 4 durability results

As can be seen from Table 4, the separation degrees between the impurities and the main peak are greater than 1.5 after the column temperature and the wavelength are changed, which indicates that the method for detecting the related substances has good durability.

EXAMPLE 3 sensitivity test

The formulation of the solution is detailed in table 5.

TABLE 5 detection limit solution preparation

Chromatographic conditions are as follows:

a chromatographic column: ODSHYPERSIL (4.6 mm. times.50 mm,3 μm);

mobile phase A: 0.05% aqueous phosphoric acid;

mobile phase B: acetonitrile-methanol (98: 2);

detection wavelength: 220 nm;

flow rate: 2.5 mL/min;

column temperature: 30 ℃;

sample introduction amount: 5 mu L of the solution;

gradient elution was performed according to the following table:

the detection data are shown in Table 6, and the map is shown in FIGS. 13-18.

TABLE 6 sensitivity test results

As can be seen from Table 6, the detection limit concentrations of travoprost, travoprost related compound A, the epoxidized derivative, the 15-epi diastereoisomer, the 5, 6-trans isomer and the 15-ketone derivative are respectively 0.019%, 0.012%, 0.0088%, 0.023% and 0.015% of the concentration of the test sample, the signal-to-noise ratio is greater than 3, the detection sensitivity is high, and the detection requirements are met.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.