CN103412082A - HPLC (High Performance Liquid Chromatography) technique-based detection method for contents of jasmonic acid compounds in lycoris radiate - Google Patents
HPLC (High Performance Liquid Chromatography) technique-based detection method for contents of jasmonic acid compounds in lycoris radiate Download PDFInfo
- Publication number
- CN103412082A CN103412082A CN2013103701314A CN201310370131A CN103412082A CN 103412082 A CN103412082 A CN 103412082A CN 2013103701314 A CN2013103701314 A CN 2013103701314A CN 201310370131 A CN201310370131 A CN 201310370131A CN 103412082 A CN103412082 A CN 103412082A
- Authority
- CN
- China
- Prior art keywords
- sample
- jasmonic
- solution
- dihydro
- jasmonic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an HPLC (High Performance Liquid Chromatography) technique-based detection method for the contents of jasmonic acid compounds in lycoris radiate. According to the detection method, multiple jasmonic acid hormones in plants can be simultaneously, quickly and accurately detected by extracting and purifying the jasmonic acid compounds in a lycoris radiate sample, establishing a standard substance separation chromatogram, and determining the contents of the jasmonic acid compounds; a detection result is reliable, and the separation degrees of various compounds are good. The detection method can be used for detecting jasmonic acid endogenous hormones in the plants and providing a technical platform for researching the roles played by various jasmonic acid hormones in growth, development and secondary metabolism processes of the plants and simultaneously researching the metabolic pathways of the jasmonic acid compounds in the plants.
Description
Technical field
The present invention relates to a kind of method of utilizing high-efficient liquid phase technique HPLC to measure hormone in plant, in particular a kind of detection method based on Jasmonates compounds content in the short-tube lycoris of HPLC technology.
Background technology
The Jasmonates compound is a parahormone that extensively is present in plant, content is very micro-in plant, the method that can be used at present detecting the Jasmonates hormone in plant mainly contains: gas chromatography mass spectrometry (GC-MS), LC-MS (HPLC-MS) and enzyme linked immunosorbent detection technology (ELISA), the methods such as UPLC-MS-MS, liquid chromatography electrochemistry detection method (liquid chromatography-electrochemical detection, HPLC-ECD) is also arranged.
In existing detection technique, independent HPLC detection method can't detect the multiple Jasmonates hormone in plant accurately and quickly.The GC-MS detection method, although sensitivity is good, need while measuring the Jasmonates compound first will carry out derivatization.The derivatization adopted is the ester derivatization, and jasmonic can be transformed into ester type compound, and endogenous Jasmonates hormone includes the compounds such as methyl jasmonate and dihydro methyl jasmonate, so the GC-MS method also has certain limitation.Euzymelinked immunosorbent assay (ELISA) detection method (ELISA) and liquid chromatography electrochemistry detection method (HPLC-ECD) have higher specificity.But the Jasmonates classes of compounds is more, the ELISA method can not detect multiple hormone simultaneously, and repeatability is poor; The HPLC-ECD method of having reported is that jasmonic is reacted with dopamine, generates the material with electrochemical signals, and the method can only detect the content of jasmonic.The step of other detection method is all complicated or to the having relatively high expectations of instrument and equipment, and is not easy to the mensuration rapidly and accurately of hormone.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of detection method based on Jasmonates compounds content in the short-tube lycoris of HPLC technology is provided, detect simultaneously comparatively rapidly and accurately four kinds of Jasmonates hormones in plant.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
(1) extraction and purification of sample
Get the fresh plant leaf 10g of testing sample, adding 20～30ml volumetric concentration is 80% methanol solution, adds adsorbent, and ice bath grinds to form homogenate, gets the supernatant rotary evaporation to water after centrifugal; Add phosphate buffer, extraction after decolouring, then redissolve, and after Spe C18 pillar purifying, uses 0.45 μ m membrane filtration, and filtrate is as sample solution;
(2) setting of pick-up unit
Chromatographic column is Eclipse XDB-C18,250mm * 4.6mm, 5 μ m; Mobile phase A methyl alcohol, the acetic acid aqueous solution of B pH3.6; Isocratic elution A:B=1:1, flow velocity are 1mL/min, detect wavelength 235nm, and column temperature is 25 ℃, treats that baseline is steady, and sample size is 10 μ L;
(3) Specification Curve of Increasing
Accurately take respectively the standard items of jasmonic JA, dihydro jasmonic DJA, methyl jasmonate MeJA and dihydro methyl jasmonate DMeJA, add methyl alcohol to dissolve, shake up and be mixed with the standard solution that mass concentration is 1.0～1.2mg/ml; Get respectively each standard solution, add the methyl alcohol stepwise dilution, make the standard serial solution that mass concentration is respectively 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 20 μ g/ml, 10 μ g/ml, 5 μ g/ml; Precision measures 10 μ l sample introductions in high performance liquid chromatograph respectively, record chromatogram, the content x of each standard items of take respectively is horizontal ordinate, the peak area y of take is ordinate, the drawing standard curve, the content of horizontal ordinate standard items is in 5～200 ug/ml, the retention time RT that obtains standard items jasmonic, dihydro jasmonic, methyl jasmonate and dihydro methyl jasmonate is respectively: jasmonic RT is 4.380min, the RT of dihydro jasmonic is 6.256min, the RT of methyl jasmonate is 8.603min, and the RT of dihydro methyl jasmonate is 13.196min;
(4) detect the Jasmonates content in testing sample short-tube lycoris tissue
The short-tube lycoris sample is carried out to the HPLC detection according to step (2) and step (3) after step (1) extraction and purification, sample introduction 10 μ l, peak area under the same retention time of integration, disposable detection obtain in short-tube lycoris sample to be measured four Jasmonates hormones and content thereof.
In described step (1), get fresh plant leaf 10g, adding 20～30ml volumetric concentration is 80% methanol solution, and 4 ℃ of lower hold over night, add cross-linking polyethylene pyrrolidone PVPP solution, and ice bath grinds to form homogenate, gets the supernatant rotary evaporation to water after centrifugal; Add phosphate buffer, and then add isopyknic sherwood oil decolouring, retain water, triplicate, regulate the pH to 8.0 of water during without color when ether, then use isopyknic ethyl acetate to extract, repeat twice, merge the ester phase, rotary evaporation, be concentrated into dry, then joining the 1ml volumetric concentration is to redissolve in 60% methyl alcohol, after Spe C18 pillar purifying, use 0.45 μ m membrane filtration, filtrate is as sample solution.
The present invention has the following advantages compared to existing technology: method of the present invention can detect multiple Jasmonates hormone simultaneously, and testing result is reliable, and various compound separation degree are better.Can be for detection of the Jasmonates endogenous hormones in plant, the effect of bringing into play in growth and development of plants and secondary metabolism process for studying various Jasmonates hormones, the study metabolic pathways for Jasmonates compound in plant provides technology platform simultaneously.
The accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of jasmonic JA of the present invention, dihydro jasmonic DJA, methyl jasmonate MeJA and dihydro methyl jasmonate DMeJA standard items;
Fig. 2 is the high-efficient liquid phase chromatogram of the present invention's short-tube lycoris sample to be measured.
Embodiment
Below embodiments of the invention are elaborated, the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The present embodiment comprises the following steps:
(1) extraction and purification of sample
Get the fresh plant leaf 10g of testing sample, adding the 25ml volumetric concentration is 80% methanol solution, and 4 ℃ of lower hold over night, add cross-linking polyethylene pyrrolidone PVPP solution, and ice bath grinds to form homogenate, gets the supernatant rotary evaporation to water after centrifugal; Add phosphate buffer, and then add isopyknic sherwood oil decolouring, retain water, triplicate, regulate the pH to 8.0 of water during without color when ether, then use isopyknic ethyl acetate to extract, repeat twice, merge the ester phase, rotary evaporation, be concentrated into dry, then joining the 1ml volumetric concentration is to redissolve in 60% methyl alcohol, after Spe C18 pillar purifying, use 0.45 μ m membrane filtration, filtrate is as sample solution;
(2) setting of pick-up unit
The HPLC device of the present embodiment: Agilent1200 high performance liquid chromatograph workstation, the Agilent1200 series pump, variable-wavelenght detector, chromatographic column is Eclipse XDB-C18,250mm * 4.6mm, 5 μ m; Mobile phase A methyl alcohol, the acetic acid aqueous solution of B pH3.6; Isocratic elution A:B=1:1, flow velocity are 1mL/min, detect wavelength 235nm, and column temperature is 25 ℃, treats that baseline is steady, and sample size is 10 μ L;
(3) Specification Curve of Increasing
Accurately take respectively the standard items of jasmonic, dihydro jasmonic, methyl jasmonate and dihydro methyl jasmonate, add methyl alcohol to dissolve, shake up and be mixed with the standard solution that mass concentration is 1.0～1.2mg/ml; Get respectively each standard solution, add the methyl alcohol stepwise dilution, make the standard serial solution that mass concentration is respectively 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 20 μ g/ml, 10 μ g/ml, 5 μ g/ml; Precision measures 10 μ l sample introductions in high performance liquid chromatograph respectively, record chromatogram, the content x of each standard items of take respectively is horizontal ordinate, the peak area y of take is ordinate, the drawing standard curve, as shown in Figure 1, in figure, JA, DJA, MeJA and DMeJA are respectively jasmonic, dihydro jasmonic, methyl jasmonate and dihydro methyl jasmonate; The content of horizontal ordinate standard items is in 5～200 ug/ml, the retention time RT that obtains standard items jasmonic, dihydro jasmonic, methyl jasmonate and dihydro methyl jasmonate is respectively: jasmonic RT is 4.380, the RT of dihydro jasmonic is 6.256, the RT of methyl jasmonate is 8.603, and the RT of dihydro methyl jasmonate is 13.196;
(4) detect the Jasmonates content in testing sample short-tube lycoris tissue
The short-tube lycoris sample is carried out to the HPLC detection according to step (2) and step (3) after step (1) extraction and purification, sample introduction 10 μ l, peak area under the same retention time of integration, disposable detection obtains in short-tube lycoris sample to be measured four Jasmonates hormones and content thereof, as shown in Figure 2, can judge according to the retention time of jasmonic, dihydro jasmonic, methyl jasmonate and dihydro methyl jasmonate, draw four Jasmonates hormones in short-tube lycoris sample to be measured, content can be judged according to peak area.
Above absolute methanol used, acetic acid and deionized water are all used 0.45 nanometer micropore membrane filtration, and absolute methanol used, acetic acid jasmonic, dihydro jasmonic, methyl jasmonate and dihydro methyl jasmonate compound purity are chromatographically pure.
Claims (2)
1. the detection method based on Jasmonates compounds content in the short-tube lycoris of HPLC technology, is characterized in that, comprises the following steps:
(1) extraction and purification of sample
Get the fresh plant leaf 10g of testing sample, adding 20～30ml volumetric concentration is 80% methanol solution, adds adsorbent, and ice bath grinds to form homogenate, gets the supernatant rotary evaporation to water after centrifugal; Add phosphate buffer, extraction after decolouring, then redissolve, and after Spe C18 pillar purifying, uses 0.45 μ m membrane filtration, and filtrate is as sample solution;
(2) setting of pick-up unit
Chromatographic column is Eclipse XDB-C18,250mm * 4.6mm, 5 μ m; Mobile phase A methyl alcohol, the acetic acid aqueous solution of B pH3.6; Isocratic elution A:B=1:1, flow velocity are 1mL/min, detect wavelength 235nm, and column temperature is 25 ℃, treats that baseline is steady, and sample size is 10 μ L;
(3) Specification Curve of Increasing
Accurately take respectively the standard items of jasmonic JA, dihydro jasmonic DJA, methyl jasmonate MeJA and dihydro methyl jasmonate DMeJA, add methyl alcohol to dissolve, shake up and be mixed with the standard solution that mass concentration is 1.0～1.2mg/ml; Get respectively each standard solution, add the methyl alcohol stepwise dilution, make the standard serial solution that mass concentration is respectively 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 20 μ g/ml, 10 μ g/ml, 5 μ g/ml; Precision measures 10 μ l sample introductions in high performance liquid chromatograph respectively, record chromatogram, the content x of each standard items of take respectively is horizontal ordinate, the peak area y of take is ordinate, the drawing standard curve, the content of horizontal ordinate standard items is in 5～200 ug/ml, the retention time RT that obtains standard items jasmonic, dihydro jasmonic, methyl jasmonate and dihydro methyl jasmonate is respectively: jasmonic RT is 4.380min, the RT of dihydro jasmonic is 6.256min, the RT of methyl jasmonate is 8.603min, and the RT of dihydro methyl jasmonate is 13.196min;
(4) detect the Jasmonates content in testing sample short-tube lycoris tissue
The short-tube lycoris sample is carried out to the HPLC detection according to step (2) and step (3) after step (1) extraction and purification, sample introduction 10 μ l, peak area under the same retention time of integration, disposable detection obtain in short-tube lycoris sample to be measured four Jasmonates hormones and content thereof.
2. a kind of detection method based on Jasmonates compounds content in the short-tube lycoris of HPLC technology according to claim 1, it is characterized in that, in described step (1), get fresh plant leaf 10g, adding 20～30ml volumetric concentration is 80% methanol solution, and 4 ℃ of lower hold over night, add cross-linking polyethylene pyrrolidone PVPP solution, ice bath grinds to form homogenate, gets the supernatant rotary evaporation to water after centrifugal; Add phosphate buffer, and then add isopyknic sherwood oil decolouring, retain water, triplicate, regulate the pH to 8.0 of water during without color when ether, then use isopyknic ethyl acetate to extract, repeat twice, merge the ester phase, rotary evaporation, be concentrated into dry, then joining the 1ml volumetric concentration is to redissolve in 60% methyl alcohol, after Spe C18 pillar purifying, use 0.45 μ m membrane filtration, filtrate is as sample solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310370131.4A CN103412082B (en) | 2013-08-22 | 2013-08-22 | HPLC (High Performance Liquid Chromatography) technique-based detection method for contents of jasmonic acid compounds in lycoris radiate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310370131.4A CN103412082B (en) | 2013-08-22 | 2013-08-22 | HPLC (High Performance Liquid Chromatography) technique-based detection method for contents of jasmonic acid compounds in lycoris radiate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103412082A true CN103412082A (en) | 2013-11-27 |
| CN103412082B CN103412082B (en) | 2014-12-24 |
Family
ID=49605108
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310370131.4A Expired - Fee Related CN103412082B (en) | 2013-08-22 | 2013-08-22 | HPLC (High Performance Liquid Chromatography) technique-based detection method for contents of jasmonic acid compounds in lycoris radiate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103412082B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297410A (en) * | 2014-11-05 | 2015-01-21 | 中国烟草总公司郑州烟草研究院 | Method for detecting abscisic acid and jasmonic acid in fresh tobacco leaves through liquid chromatogram-tandem mass spectrometry |
| CN108918710A (en) * | 2018-07-16 | 2018-11-30 | 中国烟草总公司郑州烟草研究院 | The detection method of endogenous plant hormone in a kind of fresh tobacco leaves |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004033088A (en) * | 2002-07-02 | 2004-02-05 | Japan Science & Technology Corp | Method for producing jasmonic acids and theobroxide by fungus |
| CN1596600A (en) * | 2004-08-18 | 2005-03-23 | 南京林业大学 | Long tube lycoris fast breeding method |
| CN102135528A (en) * | 2010-01-26 | 2011-07-27 | 湖南农业大学 | Detection method of jasmonic acid and methyl jasmonate in trace plant fresh sample |
-
2013
- 2013-08-22 CN CN201310370131.4A patent/CN103412082B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004033088A (en) * | 2002-07-02 | 2004-02-05 | Japan Science & Technology Corp | Method for producing jasmonic acids and theobroxide by fungus |
| CN1596600A (en) * | 2004-08-18 | 2005-03-23 | 南京林业大学 | Long tube lycoris fast breeding method |
| CN102135528A (en) * | 2010-01-26 | 2011-07-27 | 湖南农业大学 | Detection method of jasmonic acid and methyl jasmonate in trace plant fresh sample |
Non-Patent Citations (4)
| Title |
|---|
| LIU XIA 等: "Determination of both jasmonic acid and methyl jasmonate in plant samples by liquid chromatography tandem mass spectrometry", 《CHINESE SCIENCE BULLETIN》 * |
| SIBYLLE M. WILBERT 等: "Quantification of Jasmonic Acid, Methyl Jasmonate, and Salicylic Acid in Plants by Capillary Liquid Chromatography Electrospray Tandem Mass Spectrometry", 《ANALYTICAL BIOCHEMISTRY》 * |
| 王芳 等: "茉莉酸类植物激素分析研究进展", 《生命科学》 * |
| 马有宁 等: "植物内源激素预处理方法与色谱检测技术的研究进展", 《中国农学通报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297410A (en) * | 2014-11-05 | 2015-01-21 | 中国烟草总公司郑州烟草研究院 | Method for detecting abscisic acid and jasmonic acid in fresh tobacco leaves through liquid chromatogram-tandem mass spectrometry |
| CN108918710A (en) * | 2018-07-16 | 2018-11-30 | 中国烟草总公司郑州烟草研究院 | The detection method of endogenous plant hormone in a kind of fresh tobacco leaves |
| CN108918710B (en) * | 2018-07-16 | 2021-04-23 | 中国烟草总公司郑州烟草研究院 | Method for detecting endogenous phytohormones in fresh tobacco leaves |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103412082B (en) | 2014-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Thorré et al. | New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in automated microbore liquid chromatography with dual electrochemical detection | |
| CA2685358C (en) | Method of detecting blood plasma danshensu and salvianolic acid b after administration of fuzheng huayu (fzhy) | |
| Jiang et al. | Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry | |
| CN112526051B (en) | Fmoc-lysine high performance liquid chromatography determination method | |
| CN103743844B (en) | The assay method of ethanol content in methyl alcohol | |
| CN107037150A (en) | It is a kind of at the same determine food in Determination of Preservatives, sweetener and colouring agent method | |
| CN103412082B (en) | HPLC (High Performance Liquid Chromatography) technique-based detection method for contents of jasmonic acid compounds in lycoris radiate | |
| CN103630618A (en) | Method for determining betaine content in wolfberry | |
| Chen et al. | Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken | |
| CN105092720A (en) | Method for measuring genotoxic impurities in pradaxa | |
| CN103852531A (en) | Method for detecting malto-oligosaccharide in beer through HPLC-ELSD (High-Performance Liquid Chromatography-Evaporative Light Scattering Detector) | |
| CN102706971A (en) | A method for analysis of corticosteroids in cosmetics utilizing reverse microemulsion electrokinetic chromatography by taking an ionic liquid as an additive | |
| CN102854277B (en) | Determination method for matrine content in gynecological lotion | |
| CN104215721A (en) | Application of phthalic acid in amino acid analysis | |
| CN106841473B (en) | Method for rapidly analyzing content of free amino acid in fresh vegetable sample | |
| CN104237410A (en) | Detection method of substituted benzene in environmental water | |
| KR101159064B1 (en) | Quantification of active androgens in urine and blood by isotope dilution-mass spectrometry | |
| CN103776948B (en) | Method for detecting polyaspartic acid in polypeptide urea by high-performance liquid chromatography | |
| CN103217495B (en) | Method for determining turfgrass endogenous hormone | |
| CN105486788A (en) | Kit for determining zeatin nucleoside content and method thereof | |
| Mooi et al. | Simultaneous detection and quantification of zeatin and kinetin in coconut water using ultra performance liquid chromatography coupled with a simple step solid phase extraction | |
| t'Kindt et al. | Broad‐spectrum separations in metabolomics using enhanced polar LC stationary phases: A dedicated evaluation using plant metabolites | |
| CN103995062A (en) | Method for testing exenatide and impurities thereof by using high performance liquid chromatography | |
| CN103822979B (en) | The assay method of nicotine content in a kind of tobacco extract | |
| CN103983711B (en) | A kind of anoectochilus roxburghii glycosides quantitative analysis detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141224 Termination date: 20150822 |
|
| EXPY | Termination of patent right or utility model |