CN102135528A - Detection method of jasmonic acid and methyl jasmonate in trace plant fresh sample - Google Patents
Detection method of jasmonic acid and methyl jasmonate in trace plant fresh sample Download PDFInfo
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- CN102135528A CN102135528A CN2010101010623A CN201010101062A CN102135528A CN 102135528 A CN102135528 A CN 102135528A CN 2010101010623 A CN2010101010623 A CN 2010101010623A CN 201010101062 A CN201010101062 A CN 201010101062A CN 102135528 A CN102135528 A CN 102135528A
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Abstract
The invention discloses a detection method of jasmonic acid (JA) and methyl jasmonate (Me Ja) in a trace plant fresh sample on the basis of solid phase extraction and a tandem mass spectrum, comprising the following steps: (1) preparing a reverse carbon eighteen solid phase extraction small column purified and extracted by JA and MeJA; 2) extracting and purifying JA and MeJA in the plant fresh sample with a solid phase extraction method; and (3) sample detection: after the plant sample is purified and extracted, the content of JA and MeJA is detected by LCMS-MS (Liquid Chromatography Mass Spectrometry-Mass Spectrometry). The invention has the effect that the reverse carbon eighteen solid phase extraction small column is suitable for separating and purifying JA and MeJA, and JA and MeJA are simultaneously purified and separated; and meanwhile, JA and MeJA in a complex sample are separated and detected within the shorter time on the basis of the high sensitivity of the tandem mass spectrum to obtain the same result or result better than the prior detection method. The detection method has the advantages of simple result, low sample demand quantity and the like and is convenient to operate so as to detect JA and MeJA in the trace plant fresh sample at high sensitivity.
Description
Technical field
The present invention relates to assay method, is a kind of at jasmonic (JA) and methyl jasmonate (MeJA) content detecting method in the bright sample of micro-plant.
Background technology
The jasmonic compounds comprises that JA, MeJA and other derivant are the plant growth regulating substances that extensively is present in the plant.JA has obtained generally acknowledging of international academic community as a class plant hormone, expression with inducing plant defensin gene, induce the synthetic of multiple secondary metabolites, suppress photosynthesis, promote respiration, stomatal closure, leaf senile comes off and physiological function widely such as fruit maturation.Recent study shows that JA is that plant is subjected to the fastest signaling molecule of environmental stimuli afterreaction, also is the key signal molecule in the signal pathway.The research of JA has become focus in the plant at present, and the JA Determination on content then is the technical bottleneck of restriction JA research.
JA and MeJA content in plant tissue is atomic, and volatility is very strong, has increased the difficulty that it is extracted and analyzes, and requires analysis means to have very high sensitivity and selectivity.Albrecht etc. (1993) utilization euzymelinked immunosorbent assay (ELISA) has been measured the content of JA, and Baldwin etc. (1997) are with aminopropyl post purification of samples, by the GC-MS technical measurement hinder the content of JA in the inducing plant.The euzymelinked immunosorbent assay (ELISA) accuracy is relatively poor, and the aminopropyl post is not easy to be recycled, the cost height.Therefore, the extraction purifying of JA and MeJA and mensuration new technology in the micro-plant sample of urgent need employing.
Summary of the invention
The present invention be overcome in original detection technique big to the sample demand, and complex operation, detection time long deficiency, JA in the bright sample of a kind of micro-plant and the detection method of MeJA are provided.The present invention is used for the detection to bright sample JA of micro-plant and MeJA, and it is low to have an analysis cost, and analysis speed is fast, highly sensitive, and repeatability and selectivity are preferably arranged, can separate and obtain the detected peaks of JA and MeJA preferably, simplify the step of extraction and purifying simultaneously.
The technical solution adopted for the present invention to solve the technical problems mainly comprises the steps: 1) reverse carbon 18 solid-phase extraction columns of the extraction purifying of preparation JA and MeJA; 2) adopt JA and MeJA in the bright sample of extraction of solid-phase extraction column method and purifying plant; 3) sample detection: after plant sample extracted purifying, LC-MS-MS detected its JA and MeJA content.
The determined solid phase extraction of the present invention is the JA and the MeJA extraction purifying best approach in the bright sample of plant.The preparation of the solid-phase extraction column of the extraction purifying of described JA and MeJA, getting internal diameter is the SPE cylinder of 5.6mm, put into the tygon sieve plate after, add reverse carbon 18 fillers, get final product behind the balance pillar.JA and MeJA in described solid-phase extraction column method separation, the bright sample of purifying plant, sample places the centrifuge tube ice bath to grind, the methyl alcohol lixiviate that adds 600 μ L 100% is spent the night, the centrifugal 10min of 4800g, get supernatant, residual residue is with the methyl alcohol lixiviate 2h of 200 μ L 100%, the centrifugal 10min of 4800g, get supernatant, merge supernatant twice.With 3000 μ L ultrapure waters dilutions, cross solid-phase extraction column, distinguish wash-out with 200 μ L, 20% methyl alcohol and 250 μ L, 30% methyl alcohol after, with 300 μ L, 100% methanol-eluted fractions and collect jasmonic and methyl jasmonate.JA and MeJA content in the described LC-MS-MS test sample, main fragmention is m/z 133 (JA), m/z151 (MeJA), described chromatographic retention is respectively 16.27min and 17.19min.
Detection principle of the present invention: method for extraction and purification among the present invention---solid phase extraction, adopt solid-phase extraction column as separate medium, by the filling adsorption object in the pillar, adopt the mode of gradient elution, can effectively remove impurity and keep object, and sample recovery rate and reappearance are protected.Tandem mass spectrum (LC-MS-MS) detectability that the present invention adopts can reach 0.03PPb (JA) and 0.075PPb (MeJA), can detect JA extremely low in the micro-example and MeJA content, and separating effect is significantly better than HPLC, and HPLC needs the sample of more amount just can detect JA and MeJA in the sample, therefore, under the situation that is difficult to a large amount of plant samples of acquisition (as vegetable materials such as arabidopsis mutant bodies), the solid phase extraction that utilizes the present invention to set up separates and the tandem mass spectrometry detection just can solve this difficult problem.
Effect of the present invention is: utilize the present invention that the extraction refined solution of JA in the micro-plant sample and MeJA is carried out the LC-MS-MS analysis, can finish mensuration in 20min, detectability can reach 0.03PPb (JA) and 0.075PPb (MeJA), and this law is consistent with HPLC method testing result.This paper shows that to the detection of 36 routine arabidopsis mutant body materials, 127 routine paddy rice specimen materials etc. the result that this method obtains meets the requirements.It is a kind of rapid and reliable method that the tandem mass spectrum of this method introduction detects.The condition of measuring is after optimizing, and minute only needs 20min, estimates about 100 yuan of cost of determination, detects with the HPLC method and compares, and shortens 30-40min detection time, and cost is basically identical but, and step has also been simplified a lot.
Description of drawings
Fig. 1 is the comparison diagram of the different method for extraction and purification of the present invention, and A, B, C, D represent standard items respectively among the figure, ethyl acetate extraction method, dichloromethane extraction method, solid-phase extraction column method.
Fig. 2 is the comparison that tandem mass spectrum of the present invention detects JA and MeJA result in new method and the high effective liquid chromatography for measuring sample, and last figure is the tandem mass spectrum collection of illustrative plates, and figure below is that high-efficient liquid phase chromatogram is composed.
Embodiment
Below in conjunction with embodiment the present invention is further described.
One. reagent and instrument
JA, MeJA standard items (purity is 95% for Sigma, chromatographically pure), methyl alcohol (Merck KGaA, chromatographically pure), sherwood oil, ethyl acetate, hexane, methylene chloride (homemade analysis is pure), oppositely carbon 18 fillers.
Agilent 1100 high performance liquid chromatographs, the G1314A UV-detector, U.S. Thermo TSQ Quantum Ultra tandem mass spectrum system is furnished with automatic sampler, column oven and electric spray ion source, Xcalibur 2.0 data handling systems.
Two, implementation method:
The detection method of JA (JA) and MeJA (MeJA) in the bright sample of this micro-plant mainly may further comprise the steps and realizes:
1. reverse carbon 18 solid phase extraction columns that prepare the extraction purifying of JA and MeJA;
2. adopt JA and MeJA in the bright sample of extraction of solid-phase extraction column method and purifying plant;
3.LC-MS-MS JA and MeJA content in the test sample.
This method detects example:
Detection to the JA in the standard items and the MeJA recovery: pipette each 1 μ L of JA and MeJA standard items in the 1.5mL centrifuge tube with pipettor, with 100% methanol constant volume to 1mL, be mixed with the hybrid standard mother liquor, get the standard solution that an amount of hybrid standard mother liquor is mixed with 1000ng/mL, 100ng/mL, a 10ng/mL3 concentration then respectively.
The oppositely preparation of carbon 18 solid-phase extraction columns: getting internal diameter is the SPE cylinder of 5.6mm, put into the tygon sieve plate after, add reverse carbon 18 fillers, get final product behind the balance pillar.
Get two parts of the plant samples measured, a copy of it adds the JA of 100ng/mL and the mixing standard specimen of MeJA, and another part does not add standard specimen.Ice bath grinds in the 1.5mL centrifuge tube, the methyl alcohol that adds 600 μ L 100%, lixiviate is spent the night behind the mixing, the centrifugal 10min of 4800g gets supernatant in a new centrifuge tube, and residual residue is with the methyl alcohol lixiviate 2h of 200 μ L 100%, the centrifugal 10min of 4800g, get supernatant, merge supernatant twice, move in the new 5mL centrifuge tube.With 3000 μ L ultrapure waters dilutions extract, cross solid-phase extraction column, distinguish wash-out with 200 μ L, 20% methyl alcohol and 250 μ L, 30% methyl alcohol after, with 300 μ L, 100% methanol-eluted fractions and collect JA and MeJA, detect.According to the recovery of calculated by peak area JA and MeJA, the result shows: the recovery of this method reaches 92.48% (JA) and 94.3% (MeJA) respectively.
Detection to JA and MeJA in the arabidopsis mutant body material: get the bright sample 0.5000g of arabidopsis, ice bath grinds in the 1.5mL centrifuge tube, the methyl alcohol that adds 600 μ L 100%, lixiviate is spent the night behind the mixing, the centrifugal 10min of 4800g, get supernatant in a new centrifuge tube, residual residue is with the methyl alcohol lixiviate 2h of 200 μ L 100%, and the centrifugal 10min of 4800g gets supernatant, merge supernatant twice, move in the new 5mL centrifuge tube.With 3000 μ L ultrapure waters dilution extract, cross solid-phase extraction column, with behind 200 μ L, 20% methyl alcohol and 250 μ L, the 30% methyl alcohol difference wash-out (the moving phase condition sees Table 1), with 300 μ L, 100% methanol-eluted fractions and collect JA and MeJA, LC-MS-MS detects, finish in the 20min, measure for five times and be respectively 16.096 ± 2.103 and 3.443 ± 1.976 according to the JA of calculated by peak area and the concentration mean value of MeJA.The result shows that this detection method can be carried out fast detecting to JA and the MeJA in the bright sample of micro-arabidopsis.
The moving phase condition that table 1 JA and MeJA detect
Claims (4)
1. the detection method of jasmonic (JA) and methyl jasmonate (MeJA) in the micro-plant fresh sample is characterized in that may further comprise the steps:
(1) preparation JA and MeJA extract reverse carbon 18 solid-phase extraction columns of purifying;
(2) JA and MeJA in solid-phase extraction column method separation, the bright sample of purifying plant;
(3) JA and MeJA content in the LC-MS-MS test sample.
2. JA according to claim 1 and MeJA extract the preparation of reverse carbon 18 solid-phase extraction columns of purifying, it is characterized in that: adopt solid-phase extraction column.Getting internal diameter is the SPE cylinder of 5.6mm, put into the tygon sieve plate after, add reverse carbon 18 fillers, get final product behind the balance pillar.
3. JA and MeJA in solid-phase extraction column method separation according to claim 1, the bright sample of purifying plant, it is characterized in that: sample places the centrifuge tube ice bath to grind, the methyl alcohol lixiviate that adds 600 μ L 100% is spent the night, the centrifugal 10min of 4800g, get supernatant, residual residue is with the methyl alcohol lixiviate 2h of 200 μ L 100%, the centrifugal 10min of 4800g, get supernatant, merge supernatant twice.With 3000 μ L ultrapure waters dilutions, cross solid phase extraction column, distinguish wash-out with 200 μ L, 20% methyl alcohol and 250 μ L, 30% methyl alcohol after, with 300 μ L, 100% methanol-eluted fractions and collect jasmonic and methyl jasmonate.
4. JA and MeJA content in the LC-MS-MS test sample according to claim 1 is characterized in that: described main fragmention is m/z 133 (JA), m/z151 (MeJA), and described chromatographic retention is respectively 16.27min and 17.19min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103412082A (en) * | 2013-08-22 | 2013-11-27 | 安徽农业大学 | HPLC (High Performance Liquid Chromatography) technique-based detection method for contents of jasmonic acid compounds in lycoris radiate |
| CN106404962A (en) * | 2016-11-30 | 2017-02-15 | 山东省花生研究所 | Method for detecting jasmonic acid content in peanut leaves |
| CN106442791A (en) * | 2016-10-14 | 2017-02-22 | 广西壮族自治区农业科学院甘蔗研究所（中国农业科学院甘蔗研究中心） | HPLC detection method of content of jasmonic acid in sugarcane leaf |
| CN111323506A (en) * | 2020-03-23 | 2020-06-23 | 湖南农业大学 | Method for determining phytohormone in high-fat plant sample |
| CN114740109A (en) * | 2022-03-29 | 2022-07-12 | 湖南农业大学 | Method for separating and measuring methyl jasmonate enantiomer |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3916144B2 (en) * | 2002-07-02 | 2007-05-16 | 独立行政法人科学技術振興機構 | Method for producing jasmonic acids and theobroxide using microorganisms |
-
2010
- 2010-01-26 CN CN2010101010623A patent/CN102135528A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3916144B2 (en) * | 2002-07-02 | 2007-05-16 | 独立行政法人科学技術振興機構 | Method for producing jasmonic acids and theobroxide using microorganisms |
Non-Patent Citations (1)
| Title |
|---|
| XIA LIU等: "《Determination of both Jasmonic Acid(JA) and Methyl Jasmonate(MeJA) in Plant Samples by Liquid Chromatography Tandem Mass Spectrometry》", 《中国植物生理学会第十次会员代表大会暨全国学术年会论文摘要汇编—植物激素作用机理与应用》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103412082A (en) * | 2013-08-22 | 2013-11-27 | 安徽农业大学 | HPLC (High Performance Liquid Chromatography) technique-based detection method for contents of jasmonic acid compounds in lycoris radiate |
| CN106442791A (en) * | 2016-10-14 | 2017-02-22 | 广西壮族自治区农业科学院甘蔗研究所（中国农业科学院甘蔗研究中心） | HPLC detection method of content of jasmonic acid in sugarcane leaf |
| CN106442791B (en) * | 2016-10-14 | 2019-04-26 | 广西壮族自治区农业科学院甘蔗研究所（中国农业科学院甘蔗研究中心） | The HPLC detection method of jasmine acid content in a kind of Sugarcane Leaves |
| CN106404962A (en) * | 2016-11-30 | 2017-02-15 | 山东省花生研究所 | Method for detecting jasmonic acid content in peanut leaves |
| CN111323506A (en) * | 2020-03-23 | 2020-06-23 | 湖南农业大学 | Method for determining phytohormone in high-fat plant sample |
| CN111323506B (en) * | 2020-03-23 | 2023-01-17 | 湖南农业大学 | Method for determining phytohormone in high-fat plant sample |
| CN114740109A (en) * | 2022-03-29 | 2022-07-12 | 湖南农业大学 | Method for separating and measuring methyl jasmonate enantiomer |
| CN114740109B (en) * | 2022-03-29 | 2024-02-23 | 湖南农业大学 | Separation and determination method for methyl jasmonate enantiomer |
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Application publication date: 20110727 |