CN103018389B - High performance liquid chromatography method and application thereof - Google Patents

Abstract

The invention provides a high performance liquid chromatography analysis method and its application. The analysis method includes: performing component analysis on a mixture containing steroidal compounds by high performance liquid chromatography; wherein, the high performance liquid chromatography adopts reverse phase high performance liquid chromatography, and adopts gradient elution, wherein the The mobile phases of gradient elution were water and 95% acetonitrile, respectively. The method of the present invention can simultaneously analyze the mixture containing steroidal compounds (such as the mixture containing androstenedione and/or phytosterol), eliminates interference factors, greatly shortens the analysis time, is simple, reliable and accurate in operation, It has changed the previous factors such as many operation steps, long analysis time, and many interference factors, so it is an effective means to provide steroid drug research work and industrial production management of the production process.

Description

High Performance Liquid Chromatography Analysis Method and Its Application

technical field

The invention relates to the field of microbial synthesis of steroid medicines, in particular to a high-performance liquid chromatography analysis method and its application in microbial conversion of phytosterols to produce androstenedione.

Background technique

In recent years, the production of steroidal drugs has continued to grow, while the traditional production of steroidal drugs is mainly obtained by extracting diosgenin from wild Chinese medicinal materials such as "Chuan Dilong" and then chemically synthesizing it. The process is very complicated and the raw materials used are in short supply. The cost is very high, and it pollutes the environment. Therefore, the processing industry of synthetic steroid drugs using phytosterols as raw materials has risen rapidly and developed at an alarming rate. At present, phytosterols have become the main raw materials for the synthesis of steroid drugs. This fundamentally changes the shortcomings of traditional chemical synthesis of steroid drugs, such as many steps, low yield, and high price, and also makes full use of resource advantages to get rid of the constraints of raw material sources on production due to natural factors such as seasons and regions, which is conducive to protecting nature. resources and ecology.

Androstenedione (AD) is a key intermediate in the synthesis of many steroid drugs and one of the important raw materials for the production of steroid hormone drugs. As a precursor, it can continue to synthesize more than 20 kinds of steroid hormone drugs such as corticosteroids, estrogens, androgens, and anabolic hormones. The current method for producing androstenedione is to use specific microbial strains to selectively excise the side chain of phytosterols in a water/organic solvent two-phase fermentation system, thereby converting it into androstenedione. The structural formula of androstenedione is:

In the process of producing androstenedione by microbial transformation, the raw materials used are phytosterols extracted from vegetable oil refining waste and deodorized distillates, which are Brassicasterol, Campesterol, A mixture of stigmasterol and β-sitosterol. The structures of these four monomeric sterols are very similar, with only differences in individual groups. The respective structural formulas are as follows:

campesterol β-sitosterol

Campesterol β-Sitosterol

Stigmasterol Brassicasterol

Stigmasterol Brassicasterol

This general similarity in structure has brought certain difficulties to the subsequent detection process. It is difficult to separate them using traditional detection methods, so it is impossible to know which phytosterol the microorganisms are particularly fond of during the fermentation process. Raw material selection brings inconvenience. At the same time, in the fermentation process, not only androstenedione is synthesized, but also many by-products are generated, which may affect the accurate determination of androstenedione, and bring inconvenience to the production of enterprises. Therefore, it is necessary to seek one or several methods so that phytosterols and androstenedione can be accurately determined.

The methods for measuring phytosterols in the prior art are:

1. The traditional method gravimetric method, that is, digitonin is used to react with sterol under certain conditions, and a precipitate is formed to measure the sterol content after drying and weighing. This method has many steps, long analysis time and many interference factors;

2. TLC thin-layer chromatography method, and TLC thin-layer chromatography plate can only be used for rough estimation. Due to the close structure of microbial transformation products of phytosterols, the sensitivity of thin-layer chromatography method is low, which does not meet the advanced nature of production indicators;

3. The high-performance liquid chromatography detection method adopts a UV detector and uses methanol as the mobile phase. The detection wavelength is 205nm (absorption wavelength). The short wavelength has large interference and is easily affected by the mobile phase and sample processing methods. It brings certain difficulties, and the sample determination time is long, the selectivity is relatively poor, and it is difficult to detect products with similar structures;

4. For gas chromatography, the sample needs to be lipidated for 30 minutes, and it also needs to be extracted, evaporated to dryness and other operating steps. The processing time is long and there are many interference factors.

Contents of the invention

In order to solve the above-mentioned problems in the prior art, the present invention provides a high performance liquid chromatography analysis method and its application.

Specifically, the present invention provides:

(1) A high performance liquid chromatography analysis method, said analysis method comprising: carrying out component analysis to the mixture containing steroid compound with high performance liquid chromatography;

Wherein, the high-performance liquid chromatography adopts reverse-phase high-performance liquid chromatography, and adopts gradient elution, wherein the mobile phases of the gradient elution are water and 95% acetonitrile respectively.

(2) analysis method according to claim 1, wherein, said steroid compound comprises: androstenedione and/or phytosterol; Preferably, said phytosterol comprises brassicasterol, campesterol, stigmasterol and/or beta-sitosterol.

(3) The analysis method according to (1), wherein the detector used in the high performance liquid chromatography is a diode array detector (DAD).

(4) The analysis method according to (3), wherein the detection wavelength used in the high performance liquid chromatography is 210 nm.

(5) according to the analytical method described in (1), wherein, the condition of described gradient elution is set as:

0 to 6 minutes, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume;

More than 6 minutes to 30 minutes, water is 0 parts by volume, 95% acetonitrile is 100 parts by volume;

More than 30 minutes to 35 minutes, water is 10-15 parts by volume, and 95% acetonitrile is 90-85 parts by volume.

(6) according to the analytical method described in (5), wherein, the program of the time and flow rate of described gradient elution is shown in the table below:

(7) The analytical method according to (5), wherein the mobile phase flows in the A pump and the B pump respectively, and the conditions of the gradient elution are:

Wherein, the A pump is water, and the B pump is 95% acetonitrile.

(8) The analysis method according to (1), wherein the high performance liquid chromatography adopts an octadecylsilane-bonded silica gel column, and the octadecylsilane-bonded silica gel column is 150mm×4.6mm , the column temperature is 30°C, and the injection volume is 3-20 μl.

(9) According to the analysis method described in any one of (1)-(8), the analysis method further includes determining the content of each component in the mixture, and the determination includes:

1) prepare the standard solutions of androstenedione, brassicasterol, campesterol, stigmasterol and β-sitosterol of different concentrations respectively, adopt described high performance liquid chromatography to measure the peak area of described each standard solution respectively, and Draw peak area-concentration standard curves respectively for androstenedione, brassicasterol, campesterol, stigmasterol and β-sitosterol;

2) The mixture is prepared into a sample solution, and the peak area of the sample solution is determined by the high performance liquid chromatography; and

3) Calculate the concentration of each component in the sample solution from the standard curve, and thus calculate the content of each component in the mixture.

(10) The application of the analysis method described in any one of the above (1)-(9) in the production of androstenedione by microbial transformation of phytosterols, the application includes: using the analysis method to process the microbial transformation process Compositional analysis of the fermentation broth.

(11) According to the application described in (10), this application also includes: according to the result of the component analysis, determine the concentration of brassicasterol, campesterol, stigmasterol and β-sitosterol in the fermented liquid, and by This selects for the phytosterols employed in the microbial transformation.

(12) According to the application described in (10), this application also includes: according to the result of the component analysis, determine the concentration of androstenedione and/or phytosterol in the fermentation broth, and thus determine the process of production and/or end of production.

Compared with the prior art, the present invention has the following advantages and positive effects:

The invention discloses a high-performance liquid chromatography analysis method, which can be used for the central control analysis of the steroid drug production process and the production end point, and can simultaneously analyze the mixture containing steroid compounds, such as androstenedione and brassicasterol , campesterol, stigmasterol and/or β-sitosterol mixture, for analysis, eliminating interference factors, greatly shortening the analysis time, simple, reliable and accurate operation, changing the previous operation steps, long analysis time, interference There are many factors and other factors, so it is an effective means to provide steroid drug research work and industrial production management of the production process.

Description of drawings

Fig. 1 is the chromatogram of blank (acetonitrile);

Fig. 2 is the chromatogram of standard solution;

Fig. 3 is the standard curve figure of androstenedione (AD);

Fig. 4 is the standard curve figure of brassicasterol;

Fig. 5 is the standard curve figure of campesterol;

Fig. 6 is the standard curve figure of stigmasterol;

Fig. 7 is the standard curve figure of β-sitosterol;

Fig. 8 is the assay figure of the 130h fermentation product of fermentation transformation production stage;

Fig. 9 is a measurement chart of extraction, separation and purification of androstenedione (AD).

detailed description

The present invention will be further described below by the description of specific embodiment and with reference to accompanying drawing, but this is not limitation of the present invention, those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, but as long as not departing from The basic idea of the present invention is within the scope of the present invention.

The object of the present invention is to provide a high-efficiency, simple, reliable and accurate high-performance liquid chromatography central control analysis method.

In one embodiment of the analysis method of the present invention, the composition analysis is carried out with high performance liquid chromatography to the mixture containing steroidal compound (such as the mixture containing androstenedione and/or phytosterol), high performance liquid chromatography The method is reversed-phase high-performance liquid chromatography, and adopts gradient elution, and the high-performance liquid chromatography condition can be:

Chromatographic column: Octadecylsilane bonded silica gel column, that is, C ₁₈ column, 150×4.6mm (ID);

Mobile phase: A: water; B: 95% (v/v) acetonitrile.

The conditions of gradient elution can be set according to the time segment:

0 to 6 minutes, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume;

More than 6 minutes to 30 minutes, water is 0 parts by volume, 95% acetonitrile is 100 parts by volume;

More than 30 minutes to 35 minutes, water is 10-15 parts by volume, and 95% acetonitrile is 90-85 parts by volume.

For example:

0-6 minutes, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume;

6.01-30.0 minutes, water is 0 parts by volume, 95% acetonitrile is 100 parts by volume;

30.01-35.0 minutes, 10-15 parts by volume of water, and 90-85 parts by volume of 95% acetonitrile.

Further preferably, the gradient elution program shown in Table 1 is used for elution.

Table 1 Gradient elution program

Other conditions can be:

Column temperature: 30°C;

Wavelength: 210nm (DAD detector);

Injection volume: 3-20 μl (preferably 10 μl).

Drugs and medicines:

Androstenedione, reference substance (content ≥ 99%);

Brassicasterol, reference substance (content ≥ 98%);

Campesterol, reference substance (content ≥ 98%);

Stigmasterol, reference substance (content ≥ 96%);

β-sitosterol, reference substance (content ≥ 98%);

Acetonitrile (HPLC grade);

The mobile phase and solution water used in the experiment were double distilled water.

Prepare the reference substance solution of androstenedione, brassicasterol, campesterol, stigmasterol and β-sitosterol of different concentrations respectively, adopt above-mentioned high performance liquid chromatography to measure the peak area of each described reference substance solution respectively, and for Androstenedione, brassicasterol, campesterol, stigmasterol and β-sitosterol draw peak area-concentration standard curves respectively; prepare sample solution, adopt the above-mentioned high performance liquid chromatography to measure the peak area of sample solution; calculate from standard curve The concentration of each component in the sample solution.

The analysis method of the present invention can be applied in the production of androstenedione through microbial conversion of phytosterols, and samples can be collected from fermentation broth at various stages in the microbial conversion process, and then components can be analyzed by using the analysis method of the present invention. One embodiment of the application of the present invention can be: according to the results of the component analysis of the sample of the fermented liquid, determine the concentration of brassicasterol, campesterol, stigmasterol and β-sitosterol in the fermented liquid, and thus use in microbial transformation of phytosterols for selection. For example, if the consumption of stigmasterol is more than that of other phytosterols, it can be determined that the microbial strain prefers stigmasterol, so the fermentation production of the microbial strain can be optimized, for example, stigmasterol can be mainly added in the next fermentation. Another embodiment of the application of the present invention can be: according to the results of the component analysis of the sample of the fermentation broth, determine the concentration of androstenedione and/or phytosterol in the fermentation broth, and thus determine the process of production and/or production end. For example, if the concentration of androstenedione in the sample reaches a peak or the concentration of a certain phytosterol reaches a minimum value at a certain stage, it can be determined that the production is over at this time.

The content of the present invention is further explained or illustrated by means of examples below, but these examples should not be construed as limiting the protection scope of the present invention.

Experimental apparatus and chromatographic conditions in the following examples are:

Agilent 1100 high performance liquid chromatograph, Agilent 1100 DAD detector, Agilent 1100 column thermostat, chromatographic analysis records are completed by Agilent workstation.

Experimental equipment and chromatographic conditions:

Chromatographic column: C ₁₈ column, 150×4.6mm (ID);

Mobile phase: A: water; B: 95% acetonitrile;

Table 2 Gradient elution program

Column temperature: 30°C;

Wavelength: 210nm (DAD detector);

Injection volume: 10 μl.

In the following examples, androstenedione can be purchased from Chengdu Best Reagent Co., Ltd.; brassicasterol can be purchased from Supeleo company; campesterol can be purchased from Sigma company; stigmasterol can be purchased from Sigma company; Purchased from Sigma Company; acetonitrile can be purchased from Sinopharm Chemical Reagent Co., Ltd.

Embodiment 1: the selection of chromatographic conditions

1.1 Selection of mobile phase

The reason why the present invention selects acetonitrile and water as the mobile phase is for the consideration of the following factors: eliminate the interference factors of mobile relative detection during measurement to improve reliability and accuracy; improve the separation between each peak; and use gradient washing Remove androstenedione to achieve effective separation from front and rear impurities, and achieve effective separation between brassicasterol, campesterol, stigmasterol and β-sitosterol; see blank figure 1, mixed standard figure 2 (the order of peaks is androstenedione ketone (AD), brassicasterol, campesterol, stigmasterol, beta-sitosterol).

1.2 Selection of detection wavelength

The sample is dissolved in acetonitrile, and the DAD detector is used for continuous scanning at 190-400nm. It can be seen that androstenedione (AD) has a maximum absorption at 240nm; There is a maximum absorption; considering the sensitivity of each component, the detection wavelength is selected as 210nm.

1.3 Sample concentration

In order to take into account the determination of the product androstenedione (AD) and the reaction substrate brassicasterol, campesterol, stigmasterol, and β-sitosterol in the sample, the concentrations of 32.0, 204.0, 203.0, 205.0, and 203.0 μg/ml were 100%, prepare 80% and 120% concentrations according to the blank sample recovery test, and measure the test accuracy, see Table 3.

Table 3 The recovery results of androstenedione (AD) and brassicasterol, campesterol, stigmasterol and β-sitosterol

1.4 Preparation of sample solution

Accurately weigh about 0.02 g of the test product, dissolve it in acetonitrile in a 25 ml volumetric flask, ultrasonicate for 7 minutes, constant volume, shake well, and filter through an organic 0.22 μm filter membrane to obtain the test product solution.

Accurately weigh the reference substances androstenedione (AD), brassicasterol, campesterol, stigmasterol, and β-sitosterol, respectively, dissolve them in a proper amount of acetonitrile in each 25ml volumetric flask, ultrasonicate for 7 minutes, and set the volume to the standard reserve liquid.

Mixed standard solution:

Accurately pipette an appropriate amount of each standard stock solution, dilute to volume with acetonitrile in the same 25ml volumetric flask, shake well, and filter with an organic 0.22 μm filter membrane to obtain the mixed standard solution; make about androstenedione (AD) (32.0 μg/ml), brassicasterol (204.0 μg/ml), campesterol (203.0 μg/ml), stigmasterol (205.0 μg/ml), β-sitosterol (203.0 μg/ml).

Example 2

Preparation of Standard Curve

The concentrations of androstenedione (AD), brassicasterol, campesterol, stigmasterol and β-sitosterol are about 6.22-52.65μg/ml, 28.0-405.0μg/ml, 27.9-402.6μg/ml, 28.22- 407.2μg/ml and 27.93-402.9μg/ml, inject 10μl, plot the peak area A against the concentration μg/ml, see Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, the regression equation is:

Androstenedione (AD) y=6.9463x+0.12r=0.99998;

Brassicasterol y=1.8587x+1.1906r=0.99986;

Campesterol y=2.4101x+0.7561r=0.99993;

Stigmasterol y=1.0758x+1.9945r=0.99995;

β-sitosterol y=1.0767x+1.9858r=0.99993;

Example 3

Determination of standard repeatability

Mixed standard solution:

Accurately pipette an appropriate amount of each standard stock solution, dilute to volume with acetonitrile in the same 25ml volumetric flask, shake well, and filter with an organic 0.22 μm filter membrane to obtain the mixed standard solution; make about androstenedione (AD) (32.0μg/ml), brassicasterol (204.0μg/ml), campesterol (203.0μg/ml), stigmasterol (205.0μg/ml), β-sitosterol (203.0μg/ml), injection 10μl, Six needles were injected continuously, and the RSD% calculated by the peak area is shown in Table 4.

Table 4 Repeatability test result table

Example 4

Application in Fermentation Transformation Production

The fermentation broth (by weight) in the production stage of fermentation transformation comprises: sugar 10.0%, phytosterol 5.0%, vegetable oil 20%, NH ₄ NO ₃ 0.5%, pH value 8.5-8.8; add mycobacteria therein, rotating speed 250 -280rpm/min, degrade phytosterols to generate androstenedione; the growth cycle is 126-138 hours, regular sampling and testing; sampling and testing in the extraction, separation and purification stages.

Test product 1: 130-hour fermentation product in the fermentation transformation production stage

Precisely weigh about 0.5 g of the test product 1, dissolve it in a 50 ml volumetric flask with acetonitrile, ultrasonicate for 7 minutes, constant volume, shake well, and filter through an organic 0.22 μm filter membrane to obtain the test solution, injection volume: 10 μl. The result is shown in Figure 8. It can be seen that the phytosterols have been basically consumed and converted into androstenedione.

Test product 2: Extraction, isolation and purification of (androstenedione (AD)) crude product

Precisely weigh about 0.02 g of the test product 2, dissolve it in acetonitrile in a 50 ml volumetric flask, ultrasonicate for 7 minutes, constant volume, shake well, and filter through an organic 0.22 μm filter membrane to obtain the test solution, injection volume: 10 μl. The result is shown in Figure 9. Wherein it can be seen that relatively pure androstenedione (AD.

It can be seen that the high-performance liquid chromatography analysis method of the present invention can be used to control the fermentation conversion production and separation and purification process.

Claims (12)

1. A high performance liquid chromatography analysis method, said analysis method comprising: carrying out component analysis to the mixture containing steroid compound with high performance liquid chromatography; Wherein, the high-performance liquid chromatography adopts reversed-phase high-performance liquid chromatography, and adopts gradient elution, wherein the mobile phase of the gradient elution is water and 95% (v/v) acetonitrile respectively, Wherein, the high performance liquid chromatography adopts octadecylsilane bonded silica gel column; Wherein, the condition of described gradient elution is set as: 0 to 6 minutes, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume; More than 6 minutes to 30 minutes, water is 0 parts by volume, 95% acetonitrile is 100 parts by volume; More than 30 minutes to 35 minutes, water is 10-15 parts by volume, and 95% acetonitrile is 90-85 parts by volume. 2. The analysis method according to claim 1, wherein the steroid compound comprises: androstenedione and/or phytosterol. 3. The analysis method according to claim 2, wherein the phytosterol comprises brassicasterol, campesterol, stigmasterol and/or β-sitosterol. 4. The analysis method according to claim 1, wherein the detector used in the high performance liquid chromatography is a diode array detector. 5. The analysis method according to claim 4, wherein the detection wavelength adopted by the high performance liquid chromatography is 210nm. 6. analytical method according to claim 1, wherein, the time of described gradient elution and the program of flow velocity are shown in the table below: 7. analytical method according to claim 1, wherein, described mobile phase flows in A pump and B pump respectively, and the condition of described gradient elution is: Wherein, the A pump is water, and the B pump is 95% (v/v) acetonitrile. 8. The analysis method according to claim 1, wherein the octadecylsilane bonded silica gel column is 150 mm×4.6 mm, the column temperature is 30° C., and the injection volume is 3-20 μl. 9. The analysis method according to any one of claims 1-8, the analysis method further comprises measuring the content of each component in the mixture, and the determination comprises: 1) prepare the standard solutions of androstenedione, brassicasterol, campesterol, stigmasterol and β-sitosterol of different concentrations respectively, adopt described high performance liquid chromatography to measure the peak area of described each standard solution respectively, and Draw peak area-concentration standard curves respectively for androstenedione, brassicasterol, campesterol, stigmasterol and β-sitosterol; 2) The mixture is prepared into a sample solution, and the peak area of the sample solution is determined by the high performance liquid chromatography; and 3) Calculate the concentration of each component in the sample solution from the standard curve, and thus calculate the content of each component in the mixture. 10. The application of the analysis method described in any one of claims 1-9 in the production of androstenedione by phytosterol microbial conversion, the application comprises: adopting the analysis method to carry out the composition of the fermented liquid in the microbial conversion process analyze. 11. application according to claim 10, this application also comprises: according to the result of described component analysis, determine the concentration of brassicasterol, campesterol, stigmasterol and β-sitosterol in the fermented liquid, and thus The phytosterols employed in the microbial transformation are selected. 12. application according to claim 10, this application also comprises: according to the result of described component analysis, determine the concentration of androstenedione and/or phytosterol in the described fermented liquid, and thus determine the process of production and /or end of production.