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High performance liquid chromatography method and application thereof

A high-performance liquid chromatography and analysis method technology, which is applied in the high-performance liquid chromatography analysis method and in the field of phytosterol microbial transformation and production of androstenedione, can solve the problems of difficult analysis, many operation steps, long analysis time, etc. The effect of shortening analysis time, changing operating procedures, and eliminating interference factors

Inactive Publication Date: 2015-01-07
NEW FOUNDER HLDG DEV LLC +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method has many steps, long analysis time and many interference factors;
[0015] 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;
[0016] 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;
[0017] 4. Gas chromatography, the sample needs to be fattened for 30 minutes, and it needs to be extracted, evaporated to dryness and other operating steps. The processing time is long and there are many interference factors

Method used

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  • High performance liquid chromatography method and application thereof
  • High performance liquid chromatography method and application thereof
  • High performance liquid chromatography method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1: the selection of chromatographic conditions

[0096] 1.1 Selection of mobile phase

[0097] 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 To achieve effective separation of androstenedione from impurities before and after, and effective separation of brassicasterol, campesterol, stigmasterol, and β-sitosterol; see blank figure 1 , mixed standard figure 2 (The peak order is androstenedione (AD), brassicasterol, campesterol, stigmasterol, β-sitosterol).

[0098] 1.2 Selection of detection wavelength

[0099] 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 ...

Embodiment 2

[0111] Preparation of Standard Curve

[0112] 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 image 3 , Figure 4 , Figure 5 , Image 6 , Figure 7 , the regression equation is:

[0113] Androstenedione (AD) y=6.9463x+0.12r=0.99998;

[0114] Brassicasterol y=1.8587x+1.1906r=0.99986;

[0115] Campesterol y=2.4101x+0.7561r=0.99993;

[0116] Stigmasterol y=1.0758x+1.9945r=0.99995;

[0117] β-sitosterol y=1.0767x+1.9858r=0.99993;

Embodiment 3

[0119] Determination of standard repeatability

[0120] Mixed standard solution:

[0121]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.

[0122] Table 4 Repeatability test result table

[0123]

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Abstract

The invention provides a high performance liquid chromatography method and application thereof. The method is characterized by carrying out component analysis of a steroid compound-containing mixture by a high performance liquid chromatography, wherein the high performance liquid chromatography adopts reversed high performance liquid chromatography and gradient elution, and mobile phases adopted by the gradient elution respectively are water and acetonitrile having the content of 95%. Through the high performance liquid chromatography method, the steroid compound-containing mixture (such as a mixture containing androstenedione and / or phytosterol) can be simultaneously analyzed. The high performance liquid chromatography method eliminates the interference factors, greatly shortens analysis time, has simple, reliable and accurate processes, and solves the existing problems of many operation steps, long analysis time and many interference factors. Therefore, the high performance liquid chromatography method provides an effective approach for steroid drug research and production process industrial production management.

Description

technical field [0001] 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 [0002] 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/89
Inventor 岳崇斌岳光赵德
Owner NEW FOUNDER HLDG DEV LLC
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