CN103018389B - High performance liquid chromatography method and application thereof - Google Patents
High performance liquid chromatography method and application thereof Download PDFInfo
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- CN103018389B CN103018389B CN201110285805.1A CN201110285805A CN103018389B CN 103018389 B CN103018389 B CN 103018389B CN 201110285805 A CN201110285805 A CN 201110285805A CN 103018389 B CN103018389 B CN 103018389B
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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 Microbe synthesis field of steroid drugs, produce the application in androstenedione in particular to a kind of HPLC analytical method and in plant sterol microbial conversion.
Background technology
The sustainable growth of steroid drugs production in recent years, and traditional steroid drugs production forms through chemosynthesis after mainly extracting diosgenin 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, be that the processing industry of Material synthesis steroid drugs is risen rapidly with plant sterol, and grow up with surprising rapidity.Current plant sterol has become the primary raw material of steroid drugs synthesis.This fundamentally changes the deficiency of the aspects such as traditional chemical synthesizing steroid pharmaceutical procedures is many, yield is low, price; also make full use of resources advantage; break away from raw material sources because the natural cause such as season, region is to the restriction of producing, be conducive to protecting national resource and ecology.
Androstenedione (AD) is the key intermediate of many steroid drugs synthesis, is one of important raw and processed materials of producing steroid hormonal drugs.As precursor, it can continue to synthesize more than the 20 kind of steroid hormonal drugs such as all corticoids, estrogen, androgen, protein anabolic hormone.The method of current production androstenedione utilizes specific microbial strains, in water/organic solvent two-phase fermentation system, by the side chain selective ablation of plant sterol, thus changes into androstenedione.The structural formula of androstenedione is:
Produce in the process of androstenedione utilizing microbe transformation method, the raw material used is from the concise leftover bits and pieces of vegetable oil, the plant sterol extracted in deodorization distillate, it is the potpourri of brassicasterol (Brassicasterol), campesterol (Campesterol), stigmasterol (Stigmasterol), cupreol (β-Sitosterol), the structure of these 4 kinds of monomer sterols is closely similar, only there are differences on individual radical, respective structural formula is as follows:
Campesterol cupreol
Campesterol β-Sitosterol
Stigmasterol brassicasterol
Stigmasterol Brassicasterol
This structural roughly similarity brings certain difficulty to follow-up characterization processes, traditional detection method is utilized to be difficult to be separated, thus cannot learn that microorganism especially has a liking for any plant sterol during the fermentation, select to make troubles to later raw material.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 to the production of enterprise simultaneously.Therefore, be necessary to seek one or more methods, so that can Accurate Determining plant sterol and androstenedione.
The method measuring plant sterol in currently available technology has:
1. classic method gravimetric method, namely adopts digitonin to react with sterol under certain condition, generates precipitation drying and weighs and record sterol content.The method operation steps is many, analysis time is long, disturbing factor is more;
2.TLC thin layer chromatography, and "ball-park" estimate can only be carried out by TLC thin layer chromatography board, because the microbial conversion product structure of plant sterol is close, use thin layer chromatography sensitivity low, do not meet the advance of production target;
3. high performance liquid chromatography detection method, adopt UV detecting device, and using methyl alcohol as mobile phase, determined wavelength is 205nm (absorbing wavelength), and the short interference of its wavelength is large, is subject to the impact of mobile phase, sample treatment, certain difficulty is brought to analysis, and the sample determination time is longer, selectivity is relatively poor, is difficult to detect structure analogous product;
4. vapor-phase chromatography, sample need esterified 30 minutes, also need to carry out to extract, the operation steps such as evaporate to dryness, the disposal route time is long, and 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.
Specifically, the invention provides:
(1) HPLC analytical method, described analytical approach comprises: carry out constituent analysis to the potpourri high performance liquid chromatography containing steroid compound;
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 Gen Ju (1), wherein, the detecting device that described high performance liquid chromatography adopts is diode array detector (DAD).
(4) analytical approach Gen Ju (3), wherein, the determined wavelength that described high performance liquid chromatography adopts is 210nm.
(5) analytical approach Gen Ju (1), wherein, the condition of described gradient elution temporally setting stepwise is:
0 to 6 minute, water was 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume;
Be greater than 6 minutes to 30 minutes, water is 0 parts by volume, 95% acetonitrile is 100 parts by volume;
Be greater than 30 minutes to 35 minutes, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume.
(6) analytical approach Gen Ju (5), wherein, the time of described gradient elution and the program of flow velocity as shown in the table:
(7) analytical approach Gen Ju (5), wherein, described mobile phase flows respectively in A pump and B pump, and the condition of described gradient elution is:
Wherein, being water in described A pump, is 95% acetonitrile in described B pump.
(8) analytical approach Gen Ju (1), wherein, described high performance liquid chromatography adopts octadecylsilane chemically bonded silica post, and described octadecylsilane chemically bonded silica post is 150mm × 4.6mm, column temperature is 30 DEG C, and sample size is 3-20 μ l.
(9) according to analytical approach according to any one of (1)-(8), described analytical approach comprises further and measuring the content of composition each in described potpourri, and described mensuration comprises:
1) standard solution of the androstenedione of variable concentrations, brassicasterol, campesterol, stigmasterol and cupreol is prepared respectively, adopt described high performance liquid chromatography to measure the peak area of described each standard solution respectively, and draw peak area-concentration standard curve respectively for androstenedione, brassicasterol, campesterol, stigmasterol and cupreol;
2) described potpourri is mixed with sample solution, adopts the peak area of sample solution described in described high effective liquid chromatography for measuring; And
3) calculated the concentration of each composition in described sample solution by described typical curve, and calculate the content of each composition in described potpourri thus.
(10) analytical approach according to any one of above-mentioned (1)-(9) produces the application in androstenedione in plant sterol microbial conversion, and this application comprises: adopt described analytical approach to carry out constituent analysis to the fermentation liquor in this microbial conversion process.
(11) application Gen Ju (10), this application also comprises: according to the result of described constituent analysis, determine the concentration of brassicasterol, campesterol, stigmasterol and cupreol in described fermentation liquor, and thus the plant sterol adopted in described microbial conversion is selected.
(12) application Gen Ju (10), this application also comprises: according to the result of described constituent analysis, determines the concentration of androstenedione and/or plant sterol in described fermentation liquor, and determines the terminal of process and/or the production of producing thus.
Of the present inventionly compared with prior art to have the following advantages and good effect:
The invention discloses a kind of HPLC analytical method, its middle control analysis that can be used for steroid drugs production run and produce terminal, can simultaneously to the potpourri containing steroid compound, as containing androstenedione, brassicasterol, campesterol, the potpourri of stigmasterol and/or cupreol, analyze, eliminate disturbing factor, substantially reduce analysis time, simple to operate, reliably, accurately, the operation steps changed in the past is many, analysis time is long, the factors such as disturbing factor is more, therefore the effective means that steroid drugs research work and production run suitability for industrialized production manage is to provide.
Accompanying drawing explanation
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 is the mensuration figure of the 130h tunning of microbe conversion production phase;
Fig. 9 is the mensuration figure of the extraction separation and purification crude product of androstenedione (AD).
Embodiment
Below by way of embodiment description and the invention will be further described with reference to accompanying drawing, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, various amendment or improvement can be made, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
The object 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, by high performance liquid chromatography, constituent analysis is carried out to the potpourri (potpourri as containing androstenedione and/or plant sterol) containing steroid compound, high performance liquid chromatography is reversed-phase high-performance liquid chromatography, and adopt 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 temporally segmentation can be set as:
0 to 6 minute, water was 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume;
Be greater than 6 minutes to 30 minutes, water is 0 parts by volume, 95% acetonitrile is 100 parts by volume;
Be greater than 30 minutes to 35 minutes, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume.
Such as:
0-6 minute, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume;
6.01-30.0 minute, water is 0 parts by volume, 95% acetonitrile is 100 parts by volume;
30.01-35.0 minute, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume.
Preferably shown in employing table 1, gradient elution program carries out wash-out further.
Table 1 gradient elution program
Other condition can be:
Column temperature: 30 DEG C;
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);
In test, mobile phase and solution with water are redistilled water.
Prepare the reference substance solution of the androstenedione of variable concentrations, brassicasterol, campesterol, stigmasterol and cupreol respectively, adopt above-mentioned high performance liquid chromatography to measure the peak area of described each reference substance solution respectively, and draw peak area-concentration standard curve respectively for androstenedione, brassicasterol, campesterol, stigmasterol and cupreol; Preparation sample solution, adopts the peak area of above-mentioned high effective liquid chromatography for measuring sample solution; The concentration of each composition in sample solution is calculated by typical curve.
Analytical approach of the present invention can be produced in androstenedione in plant sterol microbial conversion and be applied, and can carry out collected specimens to the fermentation liquor in each stage in this microbial conversion process, then adopts analytical approach of the present invention to carry out constituent analysis.An embodiment of the present invention's application can be: according to the result of the constituent analysis of the sample of fermentation liquor, determine the concentration of brassicasterol in fermentation liquor, campesterol, stigmasterol and cupreol, and thus the plant sterol adopted in microbial conversion is selected.Such as, the consumption of stigmasterol is more than other plant sterol, then can determine this microbial strains preference stigmasterol, can be optimized so thus to the fermenting and producing of this microbial strains, such as, mainly can add stigmasterol in upper once fermentation.Another embodiment of the present invention's application can be: according to the result of the constituent analysis of the sample of fermentation liquor, determine the concentration of androstenedione and/or plant sterol in fermentation liquor, and determines the terminal of process and/or the production of producing thus.Such as, the concentration that in certain stage sample, the concentration of androstenedione reaches peak value or certain plant sterols reaches minimum, then can determine now to produce end.
Mode by the following examples further explains and describes content of the present invention, but these embodiments are not to be construed as limiting the scope of the invention.
Experimental apparatus in the examples below and chromatographic condition are:
Agilent 1100 type high performance liquid chromatograph, Agilent 1100 type DAD detecting device, Agilent 1100 type column oven, stratographic analysis record is completed by Agilent workstation.
Experimental apparatus and chromatographic condition:
Chromatographic column: C
18post, 150 × 4.6mm (ID);
Mobile phase: A: water; B:95% acetonitrile;
Table 2 gradient elution program
Column temperature: 30 DEG C;
Wavelength: 210nm (DAD detecting device);
Sample size: 10 μ l.
In the examples below, androstenedione can purchased from the special reagent company limited of Chengdu bass; Brassicasterol can purchased from Supeleo company; Campesterol can available from Sigma; Stigmasterol can available from Sigma; Cupreol can available from Sigma; Acetonitrile can purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
Embodiment 1: the selection of chromatographic condition
The selection of 1.1 mobile phases
Why the present invention selects acetonitrile and water to be mobile phase, is the consideration for following factor: when elimination measures, mobile phase is to the disturbing factor detected, to improve reliability, accuracy; Improve each peak-to-peak degree of separation; And use gradient elution that androstenedione is reached with front and back impurity to be effectively separated, to make to reach effective separation between brassicasterol, campesterol, stigmasterol, cupreol; See blank sheet 1, hybrid standard Fig. 2 (peak sequence is androstenedione (AD), brassicasterol, campesterol, stigmasterol, cupreol).
The selection of 1.2 determined wavelength
By sample dissolution in acetonitrile, carry out continuous sweep with DAD detecting device at 190-400nm, known androstenedione (AD) has absorption maximum at 240nm place; Brassicasterol, campesterol, stigmasterol, cupreol have absorption maximum at 210nm place; Take into account each component sensitivity, selection determined wavelength is 210nm.
1.3 sample concentration
In order to take into account the mensuration of product androstenedione (AD) and reaction substrate brassicasterol in sample, campesterol, stigmasterol, cupreol content, with 32.0,204.0,203.0,205.0,203.0 μ g/ml concentration for 100%, by blank application of sample recovery test preparation 80%, 120% concentration, measure its experimental accuracy, in table 3.
The recovery result of table 3 androstenedione (AD) and brassicasterol, campesterol, stigmasterol, cupreol
1.4 sample solution preparations
Precision takes test sample and is about 0.02g and dissolves in 25ml volumetric flask acetonitrile, ultrasonic 7 minutes, constant volume, shakes up, organic 0.22 μm of membrane filtration and get final product, and is need testing solution.
Precision takes reference substance androstenedione (AD), brassicasterol, campesterol, stigmasterol, cupreol respectively, dissolves with appropriate acetonitrile respectively in each 25ml volumetric flask, ultrasonic 7 minutes, and constant volume is standard reserving solution.
Hybrid standard liquid:
To pipette each standard reserving solution appropriate for precision respectively, in same 25ml volumetric flask with acetonitrile constant volume, shake up, with organic 0.22 μm of membrane filtration and get final product, be hybrid standard liquid; Make to be about 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) respectively.
Embodiment 2
The making of typical curve
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 is about respectively 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:
To pipette each standard reserving solution appropriate for precision respectively, in same 25ml volumetric flask with acetonitrile constant volume, shake up, with organic 0.22 μm of membrane filtration and get final product, be hybrid standard liquid; Make to be about 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) respectively, sample introduction 10 μ l, enter 6 pins continuously, with calculated by peak area RSD% in table 4.
Table 4 replica test result table
Embodiment 4
Application in microbe conversion production
The fermentation culture (by weight) of microbe conversion production phase comprises: sugar 10.0%, plant sterol 5.0%, vegetable oil 20%, NH
4nO
30.5%, pH value 8.5-8.8; Add mycobacterium wherein, rotating speed 250-280rpm/min, androstenedione is generated to plant sterol degraded; Growth cycle is at 126-138 hour, and timing sampling detects; The operation break-down sampling of extraction separation and purification stage is detected.
Test sample 1: the 130 hours fermentation products of microbe conversion production phase
Precision takes test sample 1 about 0.5g and dissolves in 50ml volumetric flask acetonitrile, ultrasonic 7 minutes, constant volume, shakes up, organic 0.22 μm of membrane filtration and get final product, and is need testing solution, sample size: 10 μ l.Result as shown in Figure 8.Wherein can find out, plant sterol is consumed substantially, and changes into androstenedione.
Test sample 2:(androstenedione (AD)) extraction separation and purification crude product
Precision takes test sample 2 about 0.02g and dissolves in 50ml volumetric flask acetonitrile, ultrasonic 7 minutes, constant volume, shakes up, organic 0.22 μm of membrane filtration and get final product, and is need testing solution, sample size: 10 μ l.Result as shown in Figure 9.Wherein can find out, be separated and obtained purer androstenedione (AD.
This shows, HPLC analytical method of the present invention can be adopted to control microbe conversion production and separation and purification process.
Claims (12)
1. a HPLC analytical method, described analytical approach comprises: carry out constituent analysis to the potpourri high performance liquid chromatography containing steroid compound;
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% (v/v) acetonitrile,
Wherein, described high performance liquid chromatography adopts octadecylsilane chemically bonded silica post;
Wherein, the condition of described gradient elution temporally setting stepwise be:
0 to 6 minute, water was 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume;
Be greater than 6 minutes to 30 minutes, water is 0 parts by volume, 95% acetonitrile is 100 parts by volume;
Be greater than 30 minutes to 35 minutes, water is 10-15 parts by volume, 95% acetonitrile is 90-85 parts by volume.
2. analytical approach according to claim 1, wherein, described steroid compound comprises: androstenedione and/or plant sterol.
3. analytical approach according to claim 2, wherein, described plant sterol comprises brassicasterol, campesterol, stigmasterol and/or cupreol.
4. analytical approach according to claim 1, wherein, the detecting device that described high performance liquid chromatography adopts is diode array detector.
5. analytical approach according to claim 4, wherein, the determined wavelength that described high performance liquid chromatography adopts is 210nm.
6. analytical approach according to claim 1, wherein, the time of described gradient elution and the program of flow velocity as shown in the table:
7. analytical approach according to claim 1, wherein, described mobile phase flows respectively in A pump and B pump, and the condition of described gradient elution is:
Wherein, being water in described A pump, is 95% (v/v) acetonitrile in described B pump.
8. analytical approach according to claim 1, wherein, described octadecylsilane chemically bonded silica post is 150mm × 4.6mm, and column temperature is 30 DEG C, and sample size is 3-20 μ l.
9. the analytical approach according to any one of claim 1-8, described analytical approach comprises further and measuring the content of composition each in described potpourri, and described mensuration comprises:
1) standard solution of the androstenedione of variable concentrations, brassicasterol, campesterol, stigmasterol and cupreol is prepared respectively, adopt described high performance liquid chromatography to measure the peak area of described each standard solution respectively, and draw peak area-concentration standard curve respectively for androstenedione, brassicasterol, campesterol, stigmasterol and cupreol;
2) described potpourri is mixed with sample solution, adopts the peak area of sample solution described in described high effective liquid chromatography for measuring; And
3) calculated the concentration of each composition in described sample solution by described typical curve, and calculate the content of each composition in described potpourri thus.
10. the analytical approach according to any one of claim 1-9 produces the application in androstenedione in plant sterol microbial conversion, and this application comprises: adopt described analytical approach to carry out constituent analysis to the fermentation liquor in this microbial conversion process.
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 described fermentation liquor, and thus the plant sterol adopted in described microbial conversion is selected.
12. application according to claim 10, this application also comprises: according to the result of described constituent analysis, determines the concentration of androstenedione and/or plant sterol in described fermentation liquor, and determines the terminal of process and/or the production of producing thus.
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| CN103232513B (en) * | 2013-05-09 | 2015-06-10 | 南京中医药大学 | Method for preparing tirucallol |
| 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 |
| CN115015424B (en) * | 2022-06-10 | 2023-03-21 | 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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