Full-automatic online SPE-LC-MS/MS quantitative analysis method of endogenous strigolactone in plant sample

An SPE-LC-MS, strigolactone technology, applied in the fields of biotechnology and analytical chemistry, can solve problems such as hindering the research and application of strigolactone, investing a lot of manpower and time costs, and limiting the detection sensitivity of the method. , to achieve the effect of easy data traceability, high accuracy, and improved result stability

Active Publication Date: 2019-02-22
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Application Information

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Problems solved by technology

The traditional off-line SPE technology requires manual control. Follow the above steps step by step. In the middle of the process, solvent volatilization and volume resetting must be performed, which requires a lot of manpower and time costs.
At the same time, human operation is prone to operating errors, the whole process is difficult to c

Method used

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  • Full-automatic online SPE-LC-MS/MS quantitative analysis method of endogenous strigolactone in plant sample
  • Full-automatic online SPE-LC-MS/MS quantitative analysis method of endogenous strigolactone in plant sample
  • Full-automatic online SPE-LC-MS/MS quantitative analysis method of endogenous strigolactone in plant sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, content determination of strigolactone in rice hydroponic culture fluid

[0051] 1) Material cultivation and strigolactone extraction:

[0052] The germinated rice wild type (wildtype) Shiokari was normally cultured with Kimura culture medium (amount of 100mL / plant) for 1 week, and replaced with P-deficient Kimura culture medium for P-deficiency treatment. After 3 weeks, 20 mL of the culture medium was collected and added to Stable isotope internal standard 2 h 6 -epi-5DS 500pg, add 8mL ethyl acetate, vortex for 5min, centrifuge for 5min at 2000rpm, take the upper organic phase, dry it with nitrogen, add 300μL 50% (v / v) acetonitrile / water to redissolve, enter the fully automatic online SPE-LC-MS / MS for purification, separation and detection.

[0053] 2) Online SPE process:

[0054] Hysphere for Online SPE Systems TM C18HD SPE column was purchased from Spark Holland Company. Hysphere TM The C18HD SPE column was activated with 1mL methanol and 1mL w...

Embodiment 2

[0059] The methodological parameter investigation of embodiment 2, embodiment 1 detection method

[0060] The operation was carried out according to the steps of Example 1, and the methodological parameters of the detection method were investigated.

[0061] Prepare Lepi-5DS solutions with concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 50ng / mL, and add an equivalent amount of 2ng / mL 2 h 6 -epi-5DS, detect according to the step of embodiment 1, the result shows, the ratio of the peak area of ​​epi-5DS and a certain amount of internal standard peak area and sample concentration present good linear relationship, the correlation coefficient square of linear regression equation ( R 2 ) above 0.999; the detection limit LOD (S / N=3) of this method for epi-5DS is 0.003ng / mL, and the quantification limit LOQ (S / N=10) is 0.01ng / mL. The epi-5DS analyzed in the rice phosphorus-deficient hydroponic solution is root exudate, and the intra-day and inter-day precision of the quantita...

Embodiment 3

[0064] Embodiment 3, the detection of strigolactone in the paddy rice material hydroponic fluid of different genetic background

[0065] The rice materials used in the experiment were wild type (Wildtype) and d10 and d14 mutants of Shiokari background. The operation was carried out according to the steps of Example 1, and the contents of strigolactone in the above three kinds of rice materials were detected respectively. The measured results are as image 3 As shown, the secretion of hydroponic fluid on d14 was 0.418±0.020ng / mL (n=3), and the secretion of Shiokari was 0.039±0.002ng / mL (n=3). The secretion of d14 was about 10 times that of Shiokari, and epi-5DS was not detected in the hydroponic solution of d10 roots. The d14 mutant is a dwarf mutant and an insensitive mutant of SLs, so SL will accumulate in the plant, and the secretion of epi-5DS in the hydroponic fluid should also be higher than that of the wild-type epi-5DS , the d10 mutant is a synthetic deficient mutant...

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Abstract

The invention discloses a full-automatic online SPE-LC-MS/MS quantitative analysis method of an endogenous strigolactone in plant sample. The method comprises the following steps: (1) pretreating a plant sample to obtain a treated sample; (2) dissolving the treated sample obtained in the step (1) with a redissolving solvent to obtain a solving liquid; and (3) detecting the solving liquid in an online SPE-LC-MS/MS system, recording the strigolactone and the peak area corresponding to the stable isotope interior label of the strigolactone, and calculating according to an internal standard methodto obtain the content of endogenous strigolactone in a plant sample. The method can perform accurate absolute quantitative analysis on the endogenous strigolactone of plants, is simple and convenient, has good selectivity, high accuracy and high sensitivity, and has an important meaning to the physiological function and molecular action mechanism of strigolactone compounds.

Description

technical field [0001] The invention belongs to the field of biotechnology and analytical chemistry, and in particular relates to a method for accurately and rapidly quantitatively analyzing endogenous strigolactone in plant samples by using an on-line (On-line) SPE-LC-MS / MS technology. Background technique [0002] Strigolactones (SLs) are a new class of plant hormones identified in 2008, which can play a role in the formation and growth of plant lateral roots, the elongation of tap roots and root hairs, seed germination, hypocotyl elongation and secondary growth. important role in physiological processes such as growth. In particular, strigolactone can play a key regulatory role in the establishment of plant structure by inhibiting the growth of lateral buds at extremely low concentrations, making it continue to become a hot spot and frontier in the field of plant science and agronomy research in recent years. The in-depth study of the mechanism and function of strigolact...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/045
Inventor 褚金芳夏群辛培勇
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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