阅读说明：本技术 一种抗HIV药物关键中间体β-胸苷的检测方法 (Method for detecting key intermediate β -thymidine of anti-HIV drug ) 是由 夏颖 叶婷 贾伟 郑雄敏 林崇明 于 2018-11-22 设计创作，主要内容包括：本发明提供了一种抗HIV药物关键中间体β-胸苷的检测方法,其采用高效液相色谱进行分析检测,将β-胸苷产品成品经预处理后按以下色谱条件进行检测：色谱柱：C18柱或其他等效的色谱柱；流动相：甲醇水溶液；流速：0.8ml/min-1.2ml/min；检测器：紫外检测器；检测波长为260nm-270nm；色谱柱温度：25℃-35℃；本发明快速、准确,为β-胸苷产品成品中β-胸苷的测定提供了一种可靠的分析方法,同时本发明操作简单、方便实用、安全环保、对发酵法生产β-胸苷具有现实的指导意义,且本发明的检测方法精密度好、重复性高,能够快速、准确地测定β-胸苷产品成品中β-胸苷的含量,有助于提高抗HIV药物关键中间体β-胸苷的质量和安全可控。(The invention provides a detection method of β -thymidine, a key intermediate of an anti-HIV drug, which adopts high performance liquid chromatography for analysis and detection, and detects a β -thymidine product finished product after pretreatment according to the following chromatographic conditions, wherein the chromatographic column comprises a C18 column or other equivalent chromatographic columns, a mobile phase comprises methanol aqueous solution, the flow rate is 0.8ml/min-1.2ml/min, the detector comprises an ultraviolet detector, the detection wavelength is 260nm-270nm, and the chromatographic column temperature is 25 ℃ -35 ℃.)

1. A method for detecting a key intermediate β -thymidine of an anti-HIV drug is characterized in that the content of β -thymidine and related substances are detected by high performance liquid chromatography, and the method comprises the following steps:

(1) preparing test solution by dissolving β -thymidine product with mobile phase and diluting to obtain solution containing 1mg β -thymidine per 1ml as test solution (requiring fresh preparation);

(2) preparing reference solution by precisely measuring 1ml of β -thymidine reference, placing in a 100ml measuring flask, diluting with mobile phase to 100ml scale, and shaking to obtain reference solution;

(3) β -thymidine was detected by high performance liquid chromatography:

①, detecting conditions that the detector adopted by the high performance liquid chromatography is an ultraviolet detector, the chromatographic conditions are that a chromatographic column is a C18 column or other equivalent chromatographic columns, the flow rate is 0.8ml/min-1.2ml/min, the temperature of the chromatographic column is 25 ℃ to 35 ℃, and the detection wavelength is 260nm-270 nm;

②, the detection method comprises injecting 10 μ l of reference solution into a liquid chromatograph, adjusting detection sensitivity to make the peak height of main component chromatogram peak about 20% of full range, precisely measuring 10 μ l of each of the test solution and the reference solution, injecting into the liquid chromatograph, and recording chromatogram until the retention time of the main component peak is 2.5 times.

2. The method for detecting the key intermediate β -thymidine in anti-HIV medicine according to claim 1, wherein the area of the single impurity peak in the chromatogram of the test solution is not more than 0.5 times (0.5%) of the area of the main peak in the control solution, and the sum of the areas of the impurity peaks is not more than 2 times (2.0%) of the area of the main peak in the control solution.

3. The method for detecting the key intermediate β -thymidine of the anti-HIV drug according to claim 1, wherein the method for detecting β -thymidine comprises the following steps:

(1) preparing sample, namely taking 50ml of β -thymidine product finished product, centrifuging for 10 minutes at 18000r/min, taking supernatant, measuring Xml, putting the supernatant into a volumetric flask, adding water to dissolve the supernatant and diluting the supernatant to 100 times of volume, shaking the supernatant evenly, filtering the supernatant by using a 0.45 mu m microporous membrane, taking Yml of filtrate, transferring the Yml of filtrate into the volumetric flask, adding water to dilute the supernatant to 10 times of volume, shaking the filtrate evenly, and filtering the filtrate by using the 0.45 mu m microporous membrane to obtain liquid to be detected;

(2) analyzing and detecting by adopting high performance liquid chromatography, setting six concentration gradients without zero points in the standard working solution, drawing a standard curve by taking β -thymidine as an abscissa and taking a peak area integral value as an ordinate, and calculating the content of β -thymidine in the liquid to be detected according to an external standard method to obtain the content of β -thymidine in a β -thymidine product finished product.

4. The method for detecting β -thymidine as a key intermediate of anti-HIV drug in step (3), wherein the chromatographic column in step (3) is a Shim-pack ODS C18 column of 4.6mm x 250mm and 5 μm, the flow rate is 1ml/min, the temperature of the chromatographic column is 30 ℃, and the detection wavelength is 265 nm.

5. The method for detecting the key intermediate β -thymidine of anti-HIV drugs in claim 1, wherein the mobile phase is an aqueous solution of methanol with a methanol content of 92%.

Technical Field

The invention relates to the field of β -thymidine detection, in particular to a method for detecting a key intermediate β -thymidine of an anti-HIV drug.

Background

β -thymidine is used as a key intermediate for synthesizing the zidovudine as an anti-AIDS drug, has high technical content and high price, is mainly exported to the United states, India and the like, and brings economic benefit to companies.

Disclosure of Invention

Technical problem to be solved

The invention aims to provide a method for detecting a key intermediate β -thymidine of an anti-HIV drug, which provides a quick, accurate and reliable detection and analysis method for detecting related substances in a β -thymidine product finished product and determining the content of β -thymidine, and reduces blindness.

(II) technical scheme

A method for detecting a key intermediate β -thymidine of an anti-HIV drug adopts high performance liquid chromatography to detect the content of β -thymidine and related substances, and comprises the following steps:

(1) preparing test solution by dissolving β -thymidine product with mobile phase and diluting to obtain solution containing 1mg β -thymidine per 1ml as test solution (requiring fresh preparation);

(2) preparing reference solution by precisely measuring 1ml of β -thymidine reference, placing in a 100ml measuring flask, diluting with mobile phase to 100ml scale, and shaking to obtain reference solution;

(3) β -thymidine was detected by high performance liquid chromatography:

①, detecting conditions that the detector adopted by the high performance liquid chromatography is an ultraviolet detector, the chromatographic conditions are that a chromatographic column is a C18 column or other equivalent chromatographic columns, the flow rate is 0.8ml/min-1.2ml/min, the temperature of the chromatographic column is 25 ℃ to 35 ℃, and the detection wavelength is 260nm-270 nm;

②, the detection method comprises injecting 10 μ l of reference solution into a liquid chromatograph, adjusting detection sensitivity to make the peak height of main component chromatogram peak about 20% of full range, precisely measuring 10 μ l of each of the test solution and the reference solution, injecting into the liquid chromatograph, and recording chromatogram until the retention time of the main component peak is 2.5 times.

Further, in the impurity peaks in the chromatogram of the test solution, the area of a single impurity peak is not more than 0.5 times (0.5%) of the area of the main peak of the control solution, and the sum of the areas of the impurity peaks is not more than 2 times (2.0%) of the area of the main peak of the control solution.

Further, the β -thymidine detection method specifically comprises the following steps:

(1) preparing sample, namely taking 50ml of β -thymidine product finished product, centrifuging for 10 minutes at 18000r/min, taking supernatant, measuring Xml, putting the supernatant into a volumetric flask, adding water to dissolve the supernatant and diluting the supernatant to 100 times of volume, shaking the supernatant evenly, filtering the supernatant by using a 0.45 mu m microporous membrane, taking Yml of filtrate, transferring the Yml of filtrate into the volumetric flask, adding water to dilute the supernatant to 10 times of volume, shaking the filtrate evenly, and filtering the filtrate by using the 0.45 mu m microporous membrane to obtain liquid to be detected;

(2) analyzing and detecting by adopting high performance liquid chromatography, setting six concentration gradients without zero points in the standard working solution, drawing a standard curve by taking β -thymidine as an abscissa and taking a peak area integral value as an ordinate, and calculating the content of β -thymidine in the liquid to be detected according to an external standard method to obtain the content of β -thymidine in a β -thymidine product finished product.

Further, in the step (3), the column was a 4.6mm X250 mm, 5 μm Shim-pack ODS C18 column; the flow rate is 1 ml/min; temperature of the column: 30 ℃; the detection wavelength is 265 nm;

further, the mobile phase is a methanol aqueous solution with the methanol content of 92 percent

(III) advantageous effects

Compared with the prior art, the method has the advantages of being simple in operation, convenient, practical, safe and environment-friendly, having practical guiding significance for β -thymidine production by a fermentation method, being good in precision and high in repeatability, being capable of rapidly and accurately measuring the β -thymidine content in a β -thymidine product finished product, and being beneficial to improving the quality and safety controllability of a key intermediate β -thymidine of an anti-HIV drug.

Detailed Description

The invention is further described below with reference to specific embodiments.

A method for detecting a key intermediate β -thymidine of an anti-HIV drug adopts high performance liquid chromatography to detect the content of β -thymidine and related substances, and comprises the following steps:

(1) preparing test solution by dissolving β -thymidine product with mobile phase and diluting to obtain solution containing 1mg β -thymidine per 1ml as test solution (requiring fresh preparation);

(2) preparing reference solution by precisely measuring 1ml of β -thymidine reference, placing in a 100ml measuring flask, diluting with mobile phase to 100ml scale, and shaking to obtain reference solution;

(3) β -thymidine was detected by high performance liquid chromatography:

①, detecting conditions that the detector adopted by the high performance liquid chromatography is an ultraviolet detector, the chromatographic conditions are that a chromatographic column is a C18 column or other equivalent chromatographic columns, the flow rate is 0.8ml/min-1.2ml/min, the temperature of the chromatographic column is 25 ℃ to 35 ℃, and the detection wavelength is 260nm-270 nm;

②, the detection method comprises injecting 10 μ l of reference solution into a liquid chromatograph, adjusting detection sensitivity to make the peak height of main component chromatogram peak about 20% of full range, precisely measuring 10 μ l of each of the test solution and the reference solution, injecting into the liquid chromatograph, and recording chromatogram until the retention time of the main component peak is 2.5 times.

In order to ensure the product quality, in the impurity peaks in the chromatogram of the test solution, the area of a single impurity peak is not more than 0.5 times (0.5%) of the area of a main peak of a control solution, and the sum of the areas of the impurity peaks is not more than 2 times (2.0%) of the area of the main peak of the control solution.

The detection method of β -thymidine in the β -thymidine product specifically comprises the following steps:

(1) preparing sample, namely taking 50ml of β -thymidine product finished product, centrifuging for 10 minutes at 18000r/min, taking supernatant, measuring Xml, putting the supernatant into a volumetric flask, adding water to dissolve the supernatant and diluting the supernatant to 100 times of volume, shaking the supernatant evenly, filtering the supernatant by using a 0.45 mu m microporous membrane, taking Yml of filtrate, transferring the Yml of filtrate into the volumetric flask, adding water to dilute the supernatant to 10 times of volume, shaking the filtrate evenly, and filtering the filtrate by using the 0.45 mu m microporous membrane to obtain liquid to be detected;

(2) analyzing and detecting by adopting high performance liquid chromatography, setting six concentration gradients without zero points in the standard working solution, drawing a standard curve by taking β -thymidine as an abscissa and taking a peak area integral value as an ordinate, and calculating the content of β -thymidine in the liquid to be detected according to an external standard method to obtain the content of β -thymidine in a β -thymidine product finished product.