CN104165947B - A kind of method of auxin and ABA content in quantitative assay plant - Google Patents
A kind of method of auxin and ABA content in quantitative assay plant Download PDFInfo
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Abstract
The present invention provides a kind of method of auxin and ABA content in quantitative assay plant, step is as follows: carry out lixiviate with methanol after being ground by sample, mark D5-IAA and D6-ABA in adding, EVA function concentrated solution by purification-quantitatively-concentration multiple on-line system, extract hormone with ethyl acetate equal-volume, dry up with nitrogen, then dissolve with HAc, cross Solid-Phase Extraction column purification by the SPE function in purification-quantitatively-concentration multiple on-line system, collect elution samples; Elution samples is dried, dissolve with methanol and obtain testing sample, testing sample adopts LC-MS method measure the content of auxin and abscisic acid. The present invention establishes one and saves time, analyzes method efficiently, and separating effect is desirable. Greatly reducing the consumption analyzing sample, be conducive to the simplification of operating procedure, impurity is few. The present invention realizes the automatization of pre-treatment, shortens the operating time, reduces systematic error, makes pretreatment process more accurate.
Description
Technical field
The present invention relates to a kind of method of auxin and ABA content in quantitative assay plant.
Background technology
Having multiple endogenous hormones in plant, its content and existence form are continually changing, and produce a series of biochemical reactions, to a certain extent impact and control the growth of plant, growth, the process such as ripe and old and feeble. Auxin participates in the growth of root and stem and growth, the aging of organ, the formation of vascular tissue and differentiation and development, and plant to ground and phototropic reaction etc. ABA can promote fruit and leaf abscission, organ senescence and stomatal closure, affects flowering of plant, regulate the physiological function such as growth of seed and embryo.In fruit the kind of hormone and content number directly affect the growth of fruit, hence set up reasonable and practical hormone-content and analyze method, the growth promoter of research plant is significant.
Auxin (indole-3-acetic acid in plant tissue, IAA) and abscisic acid (ABA) content very micro-, measuring difficulty, the analysis method of current plant endogenous hormones mainly has euzymelinked immunosorbent assay (ELISA) (ELISA), radioimmunology (RIA), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS) and LC-MS (LC-MS) etc. Liquid chromatograph selectivity and poor specificity, gas chromatography mass spectrometry derivatization process is loaded down with trivial details. LC-MS does not need object derivation process, easy and simple to handle, and highly sensitive, selectivity and specificity are good, but current reported liquid matter method, pre-treatment purifies only with traditional Solid phase extraction, complex operation, time and effort consuming, it is impossible to realize standardization, automatization, personal error is bigger, it is impossible to ensure accuracy and the reliability of quantitative result.
Summary of the invention
In order to solve problems of the prior art, the invention provides a kind of method of auxin and ABA content in quantitative assay plant. the present invention is on the basis of various analysis, by improving the pre-treating method in LC-MS analysis method, adopt online SPE/ gel purification/quantitative Solid-Phase Extraction (SPE) concentrating multiple on-line system and quantitatively concentrate (EVA) function, this instrument can be automatically performed SPE purification process, and EVA is by vacuum, heating, nitrogen blows the Trinity and constitutes, three road infrared sensors can precise volume setting, there is multi-solvents conversion, it is evaporated to dry, automatic dilution, the functions such as quantitative transfer, this system is common in the residual of fruit and vegerable Pesticides, the pretreatment process of plasticiser in noxious substance and food in soil and animal blood, phytohormone method for measuring has no report. IAA and ABA in testing sample is carried out Liquid Chromatography-Tandem Mass Spectrometry (high-performanceliquidchromatography-tandemmassspectrome tric, LC-MS/MS) research, adopts and add isotope (D5-IAA,D6-ABA) in sample as the means of quantitative assay, it is ensured that the reliability of qualitative, quantitative result, the research for plant endogenous hormones provides key technology method.
In order to realize foregoing invention purpose, the technical solution used in the present invention is as follows:
The invention provides a kind of method of auxin and ABA content in quantitative assay plant, step is as follows: carry out lixiviate with methanol after being ground by sample, mark D5-IAA and D6-ABA in adding, EVA function concentrated solution by purification-quantitatively-concentration multiple on-line system, extract hormone with ethyl acetate equal-volume, dry up with nitrogen, then dissolve with HAc, cross Solid-Phase Extraction column purification by the SPE function in purification-quantitatively-concentration multiple on-line system, collect elution samples; Elution samples is dried, dissolve with methanol and obtain testing sample, testing sample adopts LC-MS method measure and calculate the content of auxin and abscisic acid.
Preferably, the quality of described sample is 0.5g-2.0g.
Further, the quality of described sample is 0.5g.
Preferably, the concentration of volume percent of described methanol is 70-90%.
Further, the concentration of volume percent of described methanol is 80%.
Preferably, thickening temperature during described concentrated solution is 35-45 DEG C.
Further, thickening temperature during described concentrated solution is 35 DEG C.
Preferably, described solid-phase extraction column is C18 post.
The present invention establishes purification-quantitatively-concentration multiple on-line system liquid chromatography mass combined instrument (GPC-HPLC-MS/MS), and inner mark method ration measures the method for indole-3-acetic acid (IAA) and abscisic acid (ABA) in plant tissue. Experiment is with Semen Castaneae for material, filtering out the suitableeest extraction and purification method by orthogonal test, result shows: after testing, and draw materials 0.5g, in 4 DEG C of refrigerators of 80% methanol (pre-cooling), lixiviate is overnight, and add 500ngD6-ABA, 100ngD5-IAA35 DEG C and be concentrated into aqueous phase, extract hormone with ethyl acetate equal-volume, nitrogen is blown to do, dissolving with HAc, through the little column purification of C18, the content of ABA and IAA is of a relatively high.
The present invention by optimize two kinds of endogenous hormones of Semen Castaneae IAA, ABA extraction purification condition, establish one save time, efficient LC-MS/MS analyzes method, separating effect ideal. The only amount of samples of 0.50g, greatly reduces the consumption analyzing sample, and is conducive to the simplification of operating procedure, and impurity is few. Experiment adopts GPC purification system complete multiple step, it is achieved the automatization of pre-treatment, shorten the operating time, reduce systematic error, make pretreatment process more accurate. Endogenous hormones is very close with interior target structural formula used, thus endogenous hormones and interior mark are all synchronously performed in extraction, purge process, and both simultaneously enter chromatographic column, almost have identical retention time.
Accompanying drawing explanation
Accompanying drawing is for providing a further understanding of the present invention, and constitutes a part for description, is used for together with embodiments of the present invention explaining the present invention, is not intended that limitation of the present invention. In the accompanying drawings:
Fig. 1 is Fruits in Chinese Chestnut sample and the chromatogram of hormone standard sample; Wherein, A1 is IAA and ABA standard specimen and the total peak figure of interior target; A2 is the daughter ion peak figure of D5-IAA; A3 is the daughter ion peak figure of IAA; A4 is the daughter ion peak figure of D6-ABA; A5 is the daughter ion peak figure of ABA. B1 is total peak figure of IAA and ABA in sample; B2 is the daughter ion peak figure of D5-IAA in sample; B3 is the daughter ion peak figure of IAA in sample; B4 is the daughter ion peak figure of D6-ABA in sample; B5 is the daughter ion peak figure of ABA in sample. Herein will " B3 be the daughter ion peak figure of d5-IAA in sample; " it is revised as that " B3 is the daughter ion peak figure of IAA in sample; "
Fig. 2 is IAA and the ABA content using the method for the sample application present invention of Different Weight to detect.
Detailed description of the invention
Below example is easy to be more fully understood that the present invention, but does not limit the present invention. Experimental technique in following embodiment, if no special instructions, is conventional method.
Materials and methods
1.1 materials
Examination material, selected from swallow red Semen Castaneae (Castaneamollissima) fruit of Huairou Semen Castaneae experimental station kind matter, is put in liquid nitrogen freezing, is subsequently placed in-80 DEG C of Refrigerator stores, for hormone determination.
1.2 instruments
High performance liquid chromatography GC-MS (Agilent1200 type chromatograph of liquid Agilent6410 type series connection quadrupole rod mass spectrum); GPC(purifies-quantitatively-concentration multiple on-line system); Centrifuge (SIGMA, 3K15).
1.3 reagent
Mark (TorontoResearchChemicalsInc, D in IAA5-IAA); Mark (TorontoResearchChemicalsInc, D in ABA6-ABA); C18 post (CleanertSC18-SPE, 500mg/3mL); Anion-exchange column (CleanertSAX-SPE, 500mg/3mL);Cellulose column (AgilentBondElut-Cellulose, 500mg/3mL); Sodium diethyldithiocarbamate (antioxidant); Crospolyvinylpyrrolidone (PVPP); Methanol (chromatographically pure); Ultra-pure water provides for laboratory; Other organic reagents are analytical pure, from Beijing Chemical Plant.
The preparation of 1.4 samples
Accurately weigh the Fruits in Chinese Chestnut sample preserved in 1.1, grind, add antioxidant polyvinylpyrrolidone (PVP) and screening agent sodium diethyldithiocarbamate (BHJ) each 0.1��0.15g, adding concentration of volume percent is 80% methanol (pre-cooling), about 20 times of volumes, 4 DEG C stand overnight; Take out sample, 8000rpm, 4 DEG C of centrifugal 15min, take supernatant; It is that 80% methanol (pre-cooling) washes precipitation 3 times with 10ml concentration of volume percent, mixes supernatant; Add 100ngD5-IAA and 500ngD6-ABA, adds two ammonia, shakes up; By the EVA function of purification-quantitatively-concentration multiple on-line system, at a certain temperature solution is concentrated into 5ml; Liquid after concentration, 3500rpm, 4 DEG C of centrifugal 25min; PH to 2.5 is adjusted with 2mol/LHCl; Add a small amount of PVPP, with isopyknic extraction into ethyl acetate 3 times, collect ethyl acetate phase; Add two ammonia, mixing; By ethyl acetate N in EVA2Dry up; It is settled to 3mL with 0.1mol/LHAc; Cross solid-phase extraction column by the SPE function in purification-quantitatively-concentration multiple on-line system, collect elution samples; Adding two ammonia, vacuum lyophilization is to dry; Dissolve with 1mL methanol, obtain testing sample. Testing sample adopt LC-MS method measure and calculate the content of IAA and ABA.
Adopt Orthogonal Experiment and Design that pretreatment process is optimized, sample size is set to 2g, 1g, 0.5g; Methanol extracting concentration of volume percent is set to 90%, 80%, 70%; GPC thickening temperature is set to 45 DEG C, 40 DEG C, 35 DEG C; Solid-phase extraction column is C18, anion-exchange chromatography, cellulose column.
1.5 chromatographic conditions
Chromatographic column: ZORBAXSB-Aq post: 5 ��m, 4.6mm �� 150mm; Flow velocity: 0.3mL/min; Column temperature: 30 DEG C; Sample size: 5 �� L; Mobile phase A, containing the acetic acid aqueous solution of percent by volume 0.1%; Mobile phase B, methanol.
1.6 Mass Spectrometry Conditions
ESI ion source, negative ions detection mode, MRM pattern. Ion source condition is dry temperature 350 DEG C, dry gas stream amount 12L/min, nebulizer pressure 35psi, capillary voltage, 4000V, and optimum fragmentation voltage (frag) is 155V, and optimum collision energy (CE) is 30V.
1.7 purify-quantitatively-concentration multiple on-line system condition
1.7.1 condition is concentrated
Water heating-up temperature (Temperaturewaterheating): 35 DEG C; Concentration rifle bottom temp (Temperaturebottomcone): 35 DEG C; Rate of liquid aspiration (Suctionspeed): 20ml/min; Constant volume solution (Constantvolumesolution): ultra-pure water.
1.7.2 Solid-Phase Extraction condition
Rate of liquid aspiration (Suctionspeed): 20ml/min; Feed rate (Dispendingspeed): 20ml/min; SPE post is activated, washes post and eluting by methanol and 0.1mol/L acetic acid.
2. result and analysis
The full spectrogram (referring to Fig. 1) of 2.1 standard specimens and sample.
As it is shown in figure 1, under above chromatographic condition, IAA and ABA can be separated well with other compositions. It can be seen that standard specimen IAA, D5-IAA, ABA and D6-ABA retention time is respectively as follows: 9.089min, 9.123min, 9.514min, 9.539min.IAA, D in sample5-IAA, ABA and D6The retention time of-ABA is consistent with standard specimen. The characteristic ion of IAA and ABA, as shown in table 1.
The selection characteristic ion of table 1ABA, IAA and corresponding interior mark product
The content of IAA and ABA in 2.2 Fruits in Chinese Chestnuts
Obtaining the peak area of endogenous hormones and internal standard substance from Fig. 1, calculate the concentration of endogenous hormones ABA, IAA according to below equation, result is as shown in table 2.
In formula: the endogenous peak area of IAA/interior mark peak area=, the addition of D5-IAA is 100ng, and deuterated rate is 98%;
The endogenous peak area of ABA/interior mark peak area=, the addition of D6-ABA is 500ng, and deuterated rate is 100%.
The content obtaining IAA and ABA is calculated, as shown in table 2 by above formula.
Generally measure hormone and need substantial amounts of sample, be sometimes difficult to meet, when extracting solution consumption is certain, suitably reduce the consumption of sample so that it is more abundant that extracting solution extracts, and obtains better effect. Sample size is set to three gradients by the applicant, is divided into 0.5,1 and 2g. Applicants have discovered that the extraction ratio of methanol is high, impurity is less, employing methanol is Extraction solvent, arranges three Concentraton gradient 70%, 80% and 90%. When in vitro, phytohormone is easy to degraded or is converted into other form, control temperature critically important, in methanol concentration process, want that methanol should be removed maintenance hormone be not again destroyed, therefore in the process of concentration, have employed 35 DEG C, 40 DEG C and 45 DEG C of three temperature. In purge process, in order to reach better separating effect, have chosen the purification pillar of three types, respectively C18, anion-exchange column and cellulose column. Comprehensive above result, it has been found that: take 0.5g sample immersion extracting in 80% methanol, concentrate at 35 DEG C, the effect reached through C18 column purification is best, as shown in table 2, and use group 8 method for extraction and purification, the content of IAA and ABA is all higher, and this method is suitable for the extraction of both hormones.
IAA, ABA content in table 2 different disposal condition Fruits in Chinese Chestnut
2.3 further experiments
According to result above, the method for employing group 8, further sample size is reduced, respectively 0.50g, 0.40g, 0.20g and 0.10g, obtain result, as shown in Figure 2. When sampling amount is 0.10g and 0.20g, being not detected by IAA, the content from 0.30g to 0.50gIAA is gradually increased; From 0.10g to 0.50g, all detect that ABA and content are gradually increased. Therefore, be sampled as 0.50g, 80% methanol, C18 post and thickening temperature be 35 DEG C when extracting, IAA and ABA content is the highest.
Following example are most highly preferred embodiment of the invention:
Embodiment 1
Accurately weigh 0.5g Fruits in Chinese Chestnut sample, grind, add polyvinylpyrrolidone (PVP) and each 0.1g of BHJ, add the methanol of 80% pre-cooling, about 20 times of volumes, 4 DEG C stand overnight; Take out sample, 8000rpm, 4 DEG C of centrifugal 15min, take supernatant; Precipitate 3 times with the methanol wash column of 10ml80% pre-cooling, mix supernatant; Add 100ngD5-IAA and 500ngD6-ABA, adds two ammonia, shakes up; By the EVA function of purification-quantitatively-concentration multiple on-line system, at 35 DEG C, solution is concentrated into 5ml; Liquid after concentration, 3500rpm, 4 DEG C of centrifugal 25min; PH to 2.5 is adjusted with 2mol/LHCl; Add a small amount of PVPP, with isopyknic extraction into ethyl acetate 3 times, collect ethyl acetate phase; Add two ammonia, mixing; By ethyl acetate N in EVA2Dry up;It is settled to 3mL with 0.1mol/LHAc; Cross solid-phase extraction column C18 by the SPE function in purification-quantitatively-concentration multiple on-line system, collect elution samples; Adding two ammonia, vacuum lyophilization is to dry; Dissolve with 1mL methanol, obtain testing sample. Testing sample adopt LC-MS method measure the content of IAA and ABA.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention, although the present invention being described in detail with reference to previous embodiment, for a person skilled in the art, technical scheme described in foregoing embodiments still can be modified by it, or wherein portion of techniques feature carries out equivalent replacement. All within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (5)
1. the method for auxin and ABA content in a quantitative assay plant, it is characterised in that: step is as follows: carry out lixiviate with methanol after being ground by sample, mark D in adding5-IAA and D6-ABA, with the EVA function concentrated solution of purification-quantitatively-concentration multiple on-line system, extracts hormone with ethyl acetate equal-volume, dry up with nitrogen, dissolve with HAc again, cross Solid-Phase Extraction column purification by the SPE function in purification-quantitatively-concentration multiple on-line system, collect elution samples; Elution samples is dried, dissolve with methanol and obtain testing sample, testing sample adopts LC-MS method measure and calculate the content of auxin and abscisic acid;
Wherein, concentration condition during described concentrated solution is: water heating-up temperature: 35 DEG C; Concentration rifle bottom temp: 35 DEG C; Rate of liquid aspiration: 20ml/min; Constant volume solution: ultra-pure water;
Described solid-phase extraction column is C18 post, and extraction conditions during extraction is: rate of liquid aspiration: 20ml/min; Feed rate: 20ml/min; SPE post is activated, washes post and eluting by methanol and 0.1mol/L acetic acid;
Chromatographic condition in described LC-MS method is: chromatographic column: ZORBAXSB-Aq post: 5 ��m, 4.6mm �� 150mm; Flow velocity: 0.3mL/min; Column temperature: 30 DEG C; Sample size: 5 �� L; Mobile phase A, containing the acetic acid aqueous solution of percent by volume 0.1%; Mobile phase B, methanol;
Mass Spectrometry Conditions is: ESI ion source, negative ions detection mode, MRM pattern; Ion source condition is dry temperature 350 DEG C, dry gas stream amount 12L/min, nebulizer pressure 35psi, capillary voltage, 4000V, and optimum fragmentation voltage is 155V, and optimum collision energy is 30V.
2. method according to claim 1, it is characterised in that: the quality of described sample is 0.5g-2.0g.
3. method according to claim 2, it is characterised in that: the quality of described sample is 0.5g.
4. method according to claim 1, it is characterised in that: when described lixiviate and dissolving, the concentration of volume percent of methanol used is 70-90%.
5. method according to claim 4, it is characterised in that: when described lixiviate and dissolving, the concentration of volume percent of methanol used is 80%.
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| CN104807931B (en) * | 2015-01-04 | 2016-08-17 | 青岛农业大学 | A kind of corn germ root sheath endogenous hormones detection method |
| CN106501427A (en) * | 2016-10-26 | 2017-03-15 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and simultaneous quantitative determination of multiple endogenous plant hormones in a kind of plant sample |
| CN109212057B (en) * | 2018-08-19 | 2021-05-11 | 丁立平 | Gas chromatography-mass spectrometry combined method for measuring five trace plant growth regulators in water source water |
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