Rapid and efficient detection method of brassinosteroid
Brassinolide and detection method technology, applied in the directions of measuring devices, instruments, scientific instruments, etc., can solve the problem of not involving the determination method of brassinolide, save reagent consumption and manpower, increase extraction rate, shorten extraction effect of time
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Embodiment 1
[0023] Accurately weigh 10.00g of grape fruit, quickly grind it into powder with liquid nitrogen, add 40ml of 80% methanol and add isotope internal standard 2 H3 Brassinolide 40ng and 2 H3 brassinosterone 40ng, ultrasonically extract at low temperature for 60min, then put it in a refrigerator at 4°C for 2h, add 4ml of methanol (80%) extract to the filtered residue for 1h, filter, and combine the extracts. Use nitrogen blowing to remove the moisture in the extract solution, put the column solution through the C18 column, and carry out sample adsorption, use 40mmol / L ammonium acetate buffer solution (pH value 6.5) as the eluent to elute to obtain the adsorption solution, and pass the adsorption solution through sephadexLH -20 column for impurity removal, use methanol as the eluent to obtain the eluted product, then pass through the silica column, elute with 10ml methanol to obtain the enriched sample solution, add 5mg of 9-phenanthrene boric acid, derivatize in a 70°C water bath...
Embodiment 2
[0025] Accurately weigh 10.00g grape fruit, quickly grind it into powder with liquid nitrogen, add 40ml methanol (95%) and add isotope internal standard 2 H3 Brassinolide 40ng and 2 H3 brassinosterone 40ng, and ultrasound at low temperature for 30min, then put it in a refrigerator at 4°C for 5h, filter, add 10ml of methanol (95%) to the filter residue, put it in a refrigerator at 4°C for 1h, filter, and combine the extracts. Use nitrogen blowing to remove the moisture in the extract solution, put the column solution through the C18 column, and carry out sample adsorption, use 40mmol / L ammonium acetate buffer solution (pH value 6.5) as the eluent to elute to obtain the adsorption solution, and pass the adsorption solution through sephadexLH -20 column for impurity removal, use methanol as the eluent to obtain the eluted product, then pass through the silica column, elute with 10ml methanol to obtain the enriched sample solution, add 5mg of 9-phenanthrene boric acid, derivatize ...
Embodiment 3-6
[0027] Carry out HPLC detection to the sample to be tested of embodiment 1, concrete test condition is as follows:
[0028] Instrument and chromatographic conditions:
[0029] Instrument: Waters high performance liquid chromatography, equipped with 1525 binary HPLC pump, 717 + Autosampler, 474 fluorescence detector, Empower chromatography workstation;
[0030] Chromatographic column: C18 column (150mm×4.6mm.i.d);
[0031] Mobile phase A: double distilled water;
[0032] Mobile phase B: acetonitrile;
[0033] Flow rate: 1m / min;
[0034] Detection wavelength: the excitation wavelength is 280nm, the emission wavelength is 700nm or 310nm and 375nm are selected as the excitation wavelength and emission wavelength respectively;
[0035] Column temperature: 37°C.
[0036] Standard solution: Accurately weigh 0.0250g brassinosteroid standard substance and dilute it to 25ml with acetonitrile to obtain a standard stock solution of brassinosteroid with a concentration of 100μg / ml an...
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