CN103018389A - High performance liquid chromatography method and application thereof - Google Patents
High performance liquid chromatography method and application thereof Download PDFInfo
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- CN103018389A CN103018389A CN2011102858051A CN201110285805A CN103018389A CN 103018389 A CN103018389 A CN 103018389A CN 2011102858051 A CN2011102858051 A CN 2011102858051A CN 201110285805 A CN201110285805 A CN 201110285805A CN 103018389 A CN103018389 A CN 103018389A
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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
The present invention relates to the synthetic field of microorganism of steroid drugs, in particular to a kind of HPLC analytical method and the application in plant sterol microbial conversion production androstenedione thereof.
Background technology
In recent years steroid drugs production sustainable growth, and traditional steroid drugs production mainly is to extract diosgenin to form by chemosynthesis from the plants such as wild Chinese crude drug " earthworm ", its technique is very complicated, raw materials used in short supply, cost is very high, and contaminated environment, therefore, rise rapidly take plant sterol as the processing industry of raw material synthesizing steroid medicine, and grow up with surprising rapidity.Plant sterol has become the synthetic primary raw material of steroid drugs at present.This fundamentally changes, and traditional chemical synthesizing steroid medicine step is many, the deficiency of the aspects such as yield is low, price; also take full advantage of resources advantage; break away from the starting material source because of the restriction of the natural causes such as season, region to producing, be conducive to protect national resource and ecology.
Androstenedione (AD) is the synthetic key intermediates of many steroid drugs, is one of important raw and processed materials of producing the steroid hormone medicine.As precursor, it can continue more than the 20 kind of steroid hormone medicines such as synthetic all corticoids, estrogen, androgen, protein anabolic hormone.The method of producing at present androstenedione is to utilize specific microbial strains, in water/organic solvent two-phase fermentation system, with the side chain selective ablation of plant sterol, thereby changes into androstenedione.The structural formula of androstenedione is:
In the process of utilizing microbe transformation method production androstenedione, employed raw material is from the concise leftover bits and pieces of vegetable oil, the plant sterol that extracts in the deodorization distillate, it is brassicasterol (Brassicasterol), campesterol (Campesterol), stigmasterol (Stigmasterol), the cupreol (potpourri of β-Sitosterol), the structure of these 4 kinds of monomer sterols is closely similar, only there are differences in individual groups, structural formula separately is as follows:
The campesterol cupreol
Campesterol β-Sitosterol
The stigmasterol brassicasterol
Stigmasterol Brassicasterol
This structural roughly similarity has brought certain difficulty for follow-up characterization processes, utilize traditional detection method to be difficult to it is separated, thereby can't learn that microorganism especially has a liking for any plant sterol during the fermentation, select to make troubles for later raw material.Simultaneously during the fermentation, not only synthesized androstenedione, and had many accessory substances to generate, the generation of these accessory substances may affect the Accurate Determining of androstenedione, makes troubles for the production of enterprise.Therefore, be necessary to seek one or more methods, so that can Accurate Determining plant sterol and androstenedione.
The method of measuring at present plant sterol in the prior art has:
1. the classic method gravimetric method namely adopts digitonin to react with sterol under certain condition, and generation precipitates the drying weighing and records sterol content.The method operation steps is many, analysis time is long, disturbing factor is more;
2.TLC thin layer chromatography, and can only carry out "ball-park" estimate with the TLC thin layer chromatography board, because the microbial conversion product structure of plant sterol approaches, use thin layer chromatography sensitivity low, do not meet the advance of production target;
3. high performance liquid chromatography detection method, adopt the UV detecting device, and with methyl alcohol as mobile phase, the detection wavelength is 205nm (absorbing wavelength), and its wavelength is short to be disturbed greatly, is subject to the impact of mobile phase, sample treatment, bring certain difficulty to analysis, and the sample determination time is longer, and selectivity is relatively relatively poor, is difficult to the structural similarity product is detected;
4. vapor-phase chromatography, sample need esterified 30 minutes, also need extract, the operation steps such as evaporate to dryness, the disposal route time is long, disturbing factor is many.
Summary of the invention
For solving above-mentioned problems of the prior art, the invention provides a kind of HPLC analytical method and application thereof.
Particularly, the invention provides:
(1) a kind of HPLC analytical method, described analytical approach comprises: the potpourri that contains steroid compound is carried out constituent analysis with high performance liquid chromatography;
Wherein, described high performance liquid chromatography adopts reversed-phase high-performance liquid chromatography, and adopts gradient elution, and the mobile phase of wherein said gradient elution is respectively water and 95% acetonitrile.
(2) analytical approach according to claim 1, wherein, described steroid compound comprises: androstenedione and/or plant sterol; Preferably, described plant sterol comprises brassicasterol, campesterol, stigmasterol and/or cupreol.
(3) according to (1) described analytical approach, wherein, the detecting device that described high performance liquid chromatography adopts is diode array detector (DAD).
(4) according to (3) described analytical approach, wherein, the detection wavelength that described high performance liquid chromatography adopts is 210nm.
(5) according to (1) described analytical approach, wherein, the condition of described gradient elution is set as by the time segmentation:
0 to 6 minute, water was that 10-15 parts by volume, 95% acetonitrile are the 90-85 parts by volume;
Greater than 6 minutes to 30 minutes, water was that 0 parts by volume, 95% acetonitrile are 100 parts by volume;
Greater than 30 minutes to 35 minutes, water was that 10-15 parts by volume, 95% acetonitrile are the 90-85 parts by volume.
(6) according to (5) described analytical approach, wherein, the time of described gradient elution and the program of flow velocity are as shown in the table:
(7) according to (5) described analytical approach, wherein, described mobile phase flows in A pump and B pump respectively, and the condition of described gradient elution is:
Wherein, be water in the described A pump, be 95% acetonitrile in the described B pump.
(8) according to (1) described analytical approach, wherein, described high performance liquid chromatography adopts the octadecylsilane chemically bonded silica post, and described octadecylsilane chemically bonded silica post is 150mm * 4.6mm, and column temperature is 30 ℃, and sample size is 3-20 μ l.
(9) according to each described analytical approach in (1)-(8), described analytical approach further comprises to be measured the content of each composition in the described potpourri, and described mensuration comprises:
1) prepares respectively the standard solution of androstenedione, brassicasterol, campesterol, stigmasterol and the cupreol of variable concentrations, adopt described high performance liquid chromatography to measure respectively the peak area of described each standard solution, and draw respectively peak area-concentration standard curve for androstenedione, brassicasterol, campesterol, stigmasterol and cupreol;
2) described potpourri is mixed with sample solution, adopts the peak area of the described sample solution of described high effective liquid chromatography for measuring; And
3) calculated the concentration of each composition in the described sample solution by described typical curve, and calculate thus the content of each composition in the described potpourri.
(10) application of each described analytical approach in plant sterol microbial conversion production androstenedione in above-mentioned (1)-(9), this application comprises: adopt described analytical approach that the fermentation liquor in this microbial conversion process is carried out constituent analysis.
(11) according to (10) described application, this application also comprises: according to the result of described constituent analysis, determine the concentration of brassicasterol, campesterol, stigmasterol and cupreol in the described fermentation liquor, and thus the plant sterol that adopts in the described microbial conversion is selected.
(12) according to (10) described application, this applications also comprises: according to the result of described constituent analysis, determine the concentration of androstenedione in the described fermentation liquor and/or plant sterol, and determine thus the process of production and/or the terminal point of production.
Of the present invention compared with prior art having the following advantages and good effect:
The invention discloses a kind of HPLC analytical method, the middle control analysis that it can be used for the steroid drugs production run and produces terminal point, can be simultaneously to containing the potpourri of steroid compound, as contain androstenedione, brassicasterol, campesterol, the potpourri of stigmasterol and/or cupreol, analyze, eliminated disturbing factor, greatly shortened analysis time, simple to operate, reliably, accurately, it is many to have changed operation steps in the past, analysis time is long, therefore the factors such as disturbing factor is more provide the effective means that steroid drugs research work and production run suitability for industrialized production are managed.
Description of drawings
Fig. 1 is the chromatogram of blank (acetonitrile);
Fig. 2 is the chromatogram of standard solution;
Fig. 3 is the canonical plotting of androstenedione (AD);
Fig. 4 is the canonical plotting of brassicasterol;
Fig. 5 is the canonical plotting of campesterol;
Fig. 6 is the canonical plotting of stigmasterol;
Fig. 7 is the canonical plotting of cupreol;
Fig. 8 transforms the mensuration figure of the 130h tunning of production phase for fermentation;
Fig. 9 is the mensuration figure of the extraction separation and purification crude product of androstenedione (AD).
Embodiment
Below the invention will be further described by the description of embodiment and with reference to accompanying drawing, but this is not to be limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications or improvement, but only otherwise break away from basic thought of the present invention, all within the scope of the present invention.
The purpose of this invention is to provide a kind of efficient, simple to operate, reliably, high performance liquid chromatography middle control analysis method accurately.
In a kind of embodiment of analytical approach of the present invention, the potpourri that the contains steroid compound potpourri of androstenedione and/or plant sterol (as contain) is carried out constituent analysis with high performance liquid chromatography, high performance liquid chromatography is reversed-phase high-performance liquid chromatography, and the employing gradient elution, high-efficient liquid phase chromatogram condition can be:
Chromatographic column: octadecylsilane chemically bonded silica post, i.e. C
18Post, 150 * 4.6mm (I.D);
Mobile phase: A: water; B:95% (v/v) acetonitrile.
The condition of gradient elution can be set as by the time segmentation:
0 to 6 minute, water was that 10-15 parts by volume, 95% acetonitrile are the 90-85 parts by volume;
Greater than 6 minutes to 30 minutes, water was that 0 parts by volume, 95% acetonitrile are 100 parts by volume;
Greater than 30 minutes to 35 minutes, water was that 10-15 parts by volume, 95% acetonitrile are the 90-85 parts by volume.
For example:
0-6 minute, water was that 10-15 parts by volume, 95% acetonitrile are the 90-85 parts by volume;
6.01-30.0 minute, water is that 0 parts by volume, 95% acetonitrile are 100 parts by volume;
30.01-35.0 minute, water is that 10-15 parts by volume, 95% acetonitrile are the 90-85 parts by volume.
The gradient elution program is carried out wash-out shown in the further preferred employing table 1.
Table 1 gradient elution program
Other condition can for:
Column temperature: 30 ℃;
Wavelength: 210nm (DAD detecting device);
Sample size: 3-20 μ l (being preferably 10 μ l).
Reagent and medicine:
Androstenedione, reference substance (content 〉=99%);
Brassicasterol, reference substance (content 〉=98%);
Campesterol, reference substance (content 〉=98%);
Stigmasterol, reference substance (content 〉=96%);
Cupreol, reference substance (content 〉=98%);
Acetonitrile (HPLC level);
Mobile phase and solution with water are redistilled water in the test.
Prepare respectively the reference substance solution of androstenedione, brassicasterol, campesterol, stigmasterol and the cupreol of variable concentrations, adopt above-mentioned high performance liquid chromatography to measure respectively the peak area of described each reference substance solution, and draw respectively peak area-concentration standard curve for androstenedione, brassicasterol, campesterol, stigmasterol and cupreol; Prepare sample solution, adopt the peak area of above-mentioned high effective liquid chromatography for measuring sample solution; Calculated the concentration of each composition in the sample solution by typical curve.
Analytical approach of the present invention can be produced in the androstenedione in the plant sterol microbial conversion and be used, and can carry out collected specimens to the fermentation liquor of stages in this microbial conversion process, then adopts analytical approach of the present invention to carry out constituent analysis.The embodiment that the present invention uses can be: according to the result of the constituent analysis of the sample of fermentation liquor, determine the concentration of brassicasterol in the fermentation liquor, campesterol, stigmasterol and cupreol, and thus the plant sterol that adopts in the microbial conversion is selected.Such as, other plant sterol of the consumption rate of stigmasterol is many, then can determine this microbial strains preference stigmasterol, can the fermenting and producing of this microbial strains be optimized so thus, for example, can mainly add stigmasterol in the fermentation next time.Another embodiment that the present invention uses can be: according to the result of the constituent analysis of the sample of fermentation liquor, determine the concentration of androstenedione in the fermentation liquor and/or plant sterol, and determine thus the process of producing and/or the terminal point of production.Such as, the concentration that the concentration of androstenedione reaches peak value or certain plant sterols in certain stage sample reaches minimum, can determine then that produce this moment to finish.
Mode by the following examples further explains and describes content of the present invention, but these embodiment are not to be construed as limiting the scope of the invention.
Experimental apparatus in following examples and chromatographic condition are:
Agilent 1100 type high performance liquid chromatographs, Agilent 1100 type DAD detecting devices, Agilent 1100 type column ovens, the stratographic analysis record is finished by the Agilent workstation.
Experimental apparatus and chromatographic condition:
Chromatographic column: C
18Post, 150 * 4.6mm (ID);
Mobile phase: A: water; The B:95% acetonitrile;
Table 2 gradient elution program
Column temperature: 30 ℃;
Wavelength: 210nm (DAD detecting device);
Sample size: 10 μ l.
In following examples, androstenedione can be available from the special reagent of Chengdu bass company limited; Brassicasterol can be available from Supeleo company; Campesterol can be available from Sigma company; Stigmasterol can be available from Sigma company; Cupreol can be available from Sigma company; Acetonitrile can be available from Chemical Reagent Co., Ltd., Sinopharm Group.
Embodiment 1: the selection of chromatographic condition
1.1 the selection of mobile phase
Why the present invention selects acetonitrile and water is mobile phase, is the consideration for following factor: eliminate the disturbing factor of mobile phase to detecting when measuring, to improve reliability, accuracy; Improve each peak-to-peak degree of separation; And use gradient elution to make androstenedione and front and back impurity reach effective separation, make between brassicasterol, campesterol, stigmasterol, cupreol to reach effective separation; See blank sheet 1, hybrid standard Fig. 2 (peak sequence is androstenedione (AD), brassicasterol, campesterol, stigmasterol, cupreol).
1.2 detect the selection of wavelength
In acetonitrile, carry out continuous sweep with the DAD detecting device at 190-400nm by sample dissolution, androstenedione (AD) has absorption maximum at the 240nm place as can be known; Brassicasterol, campesterol, stigmasterol, cupreol have absorption maximum at the 210nm place; Take into account each component sensitivity, selecting to detect wavelength is 210nm.
1.3 sample concentration
In order to take into account the mensuration of product androstenedione (AD) and reaction substrate brassicasterol in the sample, campesterol, stigmasterol, cupreol content, take 32.0,204.0,203.0,205.0,203.0 μ g/ml concentration as 100%, by blank application of sample recovery test preparation 80%, 120% concentration, measure its experimental accuracy, see Table 3.
The recovery result of table 3 androstenedione (AD) and brassicasterol, campesterol, stigmasterol, cupreol
1.4 sample solution preparation
Precision takes by weighing the about 0.02g of test sample and dissolves with acetonitrile in the 25ml volumetric flask, and ultrasonic 7 minutes, constant volume, shake up, organic 0.22 μ m membrane filtration and get final product is need testing solution.
Precision takes by weighing reference substance androstenedione (AD), brassicasterol, campesterol, stigmasterol, cupreol respectively, and respectively at dissolving with an amount of acetonitrile in each 25ml volumetric flask, ultrasonic 7 minutes, constant volume was standard reserving solution.
Hybrid standard liquid:
It is an amount of that precision pipettes each standard reserving solution respectively, in same 25ml volumetric flask with the acetonitrile constant volume, shake up, with organic 0.22 μ m membrane filtration and get final product, be hybrid standard liquid; Make and be about respectively androstenedione (AD) (32.0 μ g/ml), brassicasterol (204.0 μ g/ml), campesterol (203.0 μ g/ml), stigmasterol (205.0 μ g/ml), cupreol (203.0 μ g/ml).
Embodiment 2
The making of typical curve
Be about respectively 6.22-52.65 μ g/ml, 28.0-405.0 μ g/ml, 27.9-402.6 μ g/ml, 28.22-407.2 μ g/ml and 27.93-402.9 μ g/ml in androstenedione (AD), brassicasterol, campesterol, stigmasterol and cupreol concentration, sample introduction 10 μ l, with peak area A concentration μ g/ml is mapped, see Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, 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;
Cupreol y=1.0767x+1.9858r=0.99993;
Embodiment 3
The mensuration of standard weight renaturation
Hybrid standard liquid:
It is an amount of that precision pipettes each standard reserving solution respectively, in same 25ml volumetric flask with the acetonitrile constant volume, shake up, with organic 0.22 μ m membrane filtration and get final product, be hybrid standard liquid; Make and be about respectively androstenedione (AD) (32.0 μ g/ml), brassicasterol (204.0 μ g/ml), campesterol (203.0 μ g/ml), stigmasterol (205.0 μ g/ml), cupreol (203.0 μ g/ml), sample introduction 10 μ l, advance continuously 6 pins, RSD% sees Table 4 with calculated by peak area.
Table 4 replica test is table as a result
Embodiment 4
Fermentation transforms the application in producing
The fermentation culture (by weight) that fermentation transforms the production phase comprises: sugar 10.0%, plant sterol 5.0%, vegetable oil 20%, NH
4NO
30.5%, pH value 8.5-8.8; To wherein adding mycobacterium, rotating speed 250-280rpm/min is to plant sterol degraded generation androstenedione; Growth cycle was at 126-138 hour, and timing sampling detects; The operation break-down sampling of extraction separation and purification stage is detected.
Test sample 1: fermentation transforms 130 hours tunnings of production phase
Precision takes by weighing test sample 1 about 0.5g and dissolves with acetonitrile in the 50ml volumetric flask, and ultrasonic 7 minutes, constant volume, shake up, organic 0.22 μ m membrane filtration and get final product is need testing solution, sample size: 10 μ l.The result as shown in Figure 8.Can find out that wherein plant sterol is consumed substantially, and changes into androstenedione.
Test sample 2:(androstenedione (AD)) extraction separation and purification crude product
Precision takes by weighing test sample 2 about 0.02g and dissolves with acetonitrile in the 50ml volumetric flask, and ultrasonic 7 minutes, constant volume, shake up, organic 0.22 μ m membrane filtration and get final product is need testing solution, sample size: 10 μ l.The result as shown in Figure 9.Wherein can find out, separate obtaining purer androstenedione (AD.
This shows, can adopt HPLC analytical method of the present invention that fermentation conversion production and separation and purification process are controlled.
Claims (12)
1. HPLC analytical method, described analytical approach comprises: the potpourri that contains steroid compound is carried out constituent analysis with high performance liquid chromatography;
Wherein, described high performance liquid chromatography adopts reversed-phase high-performance liquid chromatography, and adopts gradient elution, and the mobile phase of wherein said gradient elution is respectively water and 95% acetonitrile.
2. analytical approach according to claim 1, wherein, described steroid compound comprises: androstenedione and/or plant sterol; Preferably, described plant sterol comprises brassicasterol, campesterol, stigmasterol and/or cupreol.
3. analytical approach according to claim 1, wherein, the detecting device that described high performance liquid chromatography adopts is diode array detector.
4. analytical approach according to claim 3, wherein, the detection wavelength that described high performance liquid chromatography adopts is 210nm.
5. analytical approach according to claim 1, wherein, the condition of described gradient elution is set as by the time segmentation:
0 to 6 minute, water was that 10-15 parts by volume, 95% acetonitrile are the 90-85 parts by volume;
Greater than 6 minutes to 30 minutes, water was that 0 parts by volume, 95% acetonitrile are 100 parts by volume;
Greater than 30 minutes to 35 minutes, water was that 10-15 parts by volume, 95% acetonitrile are the 90-85 parts by volume.
6. analytical approach according to claim 5, wherein, the time of described gradient elution and the program of flow velocity are as shown in the table:
7. analytical approach according to claim 5, wherein, described mobile phase flows in A pump and B pump respectively, and the condition of described gradient elution is:
Wherein, be water in the described A pump, be 95% acetonitrile in the described B pump.
8. analytical approach according to claim 1, wherein, described high performance liquid chromatography adopts the octadecylsilane chemically bonded silica post, and described octadecylsilane chemically bonded silica post is 150mm * 4.6mm, and column temperature is 30 ℃, and sample size is 3-20 μ l.
9. each described analytical approach according to claim 1-8, described analytical approach further comprise to be measured the content of each composition in the described potpourri, and described mensuration comprises:
1) prepares respectively the standard solution of androstenedione, brassicasterol, campesterol, stigmasterol and the cupreol of variable concentrations, adopt described high performance liquid chromatography to measure respectively the peak area of described each standard solution, and draw respectively peak area-concentration standard curve for androstenedione, brassicasterol, campesterol, stigmasterol and cupreol;
2) described potpourri is mixed with sample solution, adopts the peak area of the described sample solution of described high effective liquid chromatography for measuring; And
3) calculated the concentration of each composition in the described sample solution by described typical curve, and calculate thus the content of each composition in the described potpourri.
10. the application of each described analytical approach in plant sterol microbial conversion production androstenedione among the claim 1-9, this application comprises: adopt described analytical approach that the fermentation liquor in this microbial conversion process is carried out constituent analysis.
11. application according to claim 10, this application also comprises: according to the result of described constituent analysis, determine the concentration of brassicasterol, campesterol, stigmasterol and cupreol in the described fermentation liquor, and thus the plant sterol that adopts in the described microbial conversion is selected.
12. application according to claim 10, this application also comprises: according to the result of described constituent analysis, determine the concentration of androstenedione in the described fermentation liquor and/or plant sterol, and determine thus the process of producing and/or the terminal point of production.
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| CN107782828A (en) * | 2017-12-12 | 2018-03-09 | 江南大学 | A kind of method by high efficiency liquid chromatography for separating and determining campesterol, stigmasterol and Sitosterolum |
| CN115015424A (en) * | 2022-06-10 | 2022-09-06 | 完美（广东）日用品有限公司 | High performance liquid chromatography fingerprint construction method and application thereof |
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| CN115015424A (en) * | 2022-06-10 | 2022-09-06 | 完美（广东）日用品有限公司 | High performance liquid chromatography fingerprint construction method and application thereof |
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