CN102980953B - Method for quantitative detection of endogenous brassinosteroids in plant sample - Google Patents
Method for quantitative detection of endogenous brassinosteroids in plant sample Download PDFInfo
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Abstract
The invention discloses a method for quantitative detection of endogenous brassinosteroids in a plant sample. According to the method, firstly the endogenous brassinosteroids are extracted by using a solvent, solid-phase extraction impurity removal is carried out by double-layer solid-phase extraction small columns, liquid-liquid extraction impurity removal is further carried out, and then, a high-performance liquid-electrospray-tandem mass spectrometry is adopted, so that the quantitative detection of the endogenous brassinosteroids is realized. The method disclosed by the invention has the advantages that the operation is simple and fast, and the sample consumption is little; and meanwhile, most of plant matrixes are effectively removed, thus the quantitative detection of low-content endogenous brassinosteroids can be realized.
Description
Technical field
The quantitative detecting method that the present invention relates to endogenous rape element sterol in a kind of plant sample, belongs to analytical chemistry field.
Background technology
Rape element sterol (brassinosteroids, BRs) is a kind of polyhydroxylated phytosterin compound, is the 6th class plant hormone of finding after auxin, gibberellin, the basic element of cell division, abscisic acid and ethene.Up to now, have been found that and comprise 28-norbrassinolide, 28-norcastasterone, brassinosteroid (brassinolide), 24-epi-brassinolide (24-epibrassinolide), rape element sterone (castasterone), 24-table rape element sterone (24-epicastasterone), tens kinds of BRs compounds such as high rape plain lactone (homobrassinolide).BRs is extremely low at plant intensive amount, is generally 0.01-1ng/g.Rape element sterol is regulating and controlling a series of physiology and the metabolic process of plant, comprises the germination of seed, seedling grow into maturation, the growth of seed etc.Simultaneously, BRs is the elongation to vegetable cell, division and differentiation also, the increase of crop yield, reproduction, aging, to the induction of Synthesis pathway, the advolution of root, the formation of pollen tube, the activation of proton pump, the specificity physiological processes such as photosynthesis and the perception to antioxidant system play an important role.In recent years, about the physiology function of rape element sterol, the biosynthesizing of for example BRs, the research of degraded and metabolic pathway is subject to extensive concern.But because extract mesostroma complexity and the rape element sterol content of plant are extremely low, carrying out of these researchs is subject to certain limitation.Therefore, set up efficient sample-pretreating method and in conjunction with highly sensitive detection method be BRs detect key.
At present, in the plant of having reported, the pre-treating method of rape element sterol comprises liquid-liquid extraction, Solid-Phase Extraction, magnetic Solid-Phase Extraction and liquid chromatography purification process etc.These methods all propose very high requirement to sample size, and more than majority requires 40g fresh weight, obvious so large sample size cannot realize for the precious sample of part, also brings solvent consumption greatly and high in cost of production problem to sample pretreatment process simultaneously.
Summary of the invention
Technical matters to be solved by this invention is to overcome the deficiencies in the prior art, provide a kind of simply, the disposal route of plant sample fast, and realize the quantitative detection of endogenous rape element sterol.
The present invention for solving the problems of the technologies described above proposed technical scheme is: the quantitative detecting method of endogenous rape element sterol in a kind of plant sample, comprises the following steps:
1) extract endogenous rape element sterol: after accurately weighing plant sample, be placed in mortar, liquid nitrogen frozen be ground to Powdered after, add Isotopic Internal Standard
2h
3brassinosteroid and
2h
3rape element sterone, after extracting with acetonitrile, more than-18 DEG C of following placement 12h;
2) dewater: to step 1) extract after sample dewater;
3) Solid-Phase Extraction removal of impurities: by step 2) sample solution after the dewatering double-deck solid phase extraction column after by activation, after desorb, collect loading efflux and stripping liquid, remove the solvent in sample;
4) liquid-liquid extraction removal of impurities: by step 3) sample of gained is with ether dissolution, and vortex is abandoned water after leaving standstill, and adding pH is 2～10 buffer solution, and vortex is abandoned water after leaving standstill, and repeats removal of impurities more than 3 times or 3 times, gets ether phase, dries up constant volume;
5) constant volume detects: by step 4) sample after constant volume enters high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, measures rape element sterol.
The filler of above-mentioned double-deck solid phase extraction column is ketjenblack EC/ethylenediamine-N-propyl group bonded silica gel.
Dewater is by step 1) sample after extracting is centrifugal, gets supernatant, in supernatant, adds sodium chloride, makes water and organic phase layering with vortex mixed instrument concuss, and abandon water, then add anhydrous magnesium sulfate to dewater in organic phase.
Buffer solution is the aqueous solution with buffer salt preparations such as sodium hydrogen phosphate, ammonium formates.
Plant sample of the present invention comprises: tissue and the tissue extract thereof such as the roots of the plants such as paddy rice, rape, arabidopsis, tomato, tobacco leaf, stem, leaf, flower, pollen.
In Solid-Phase Extraction step, ethylenediamine-N-propyl group bonded silica gel is the most frequently used adsorbent, and it such as, shows good performance at aspects such as removing polar impurity (organic acid, polarity pigment, carbohydrate).In addition, the hydrophobic matrix in plant substrates also has certain influence to follow-up liquid phase separation and sensitivity of mass spectrometry.Ketjenblack EC has very strong hydrophobicity, and the compound of energy and π system produces π-π effect.Chlorophyll in plant extraction liquid is the π architecture compound of plane, is expected to be removed by π-π effect combination with ketjenblack EC.Therefore, ketjenblack EC and ethylenediamine-N-propyl group bonded silica gel are in conjunction with can effectively removing polarity and the non polar impurities in plant extraction liquid.
The present invention can remove plant substrates effectively with double-deck solid-phase extraction column (ketjenblack EC/ethylenediamine-N-propyl group bonded silica gel), the sample size needing is little, realize rape element sterol detection method fast and effectively simultaneously, only spend the middle detection that realizes endogenous rape element sterol at 1g plant leaf blade and 0.5g.
Brief description of the drawings
Fig. 1 is the multiple-reaction monitoring figure of the standard model of 7 kinds of rape element sterols, wherein, 1 is the standard model of 28-norbrassinolide, 2 is the standard model of 28-norcastasterone, 3 is the standard model of brassinolide, 4 standard models that are 24-epibrassinolide, 5 standard models that are castasterone, 6 is the standard model of 24-epicastasterone, 7 standard models that are homobrassinolide.
Fig. 2 is the multiple-reaction monitoring figure of application endogenous rape element provided by the present invention sterol, wherein A
1and A
2it is the rape leave sample in embodiment 5; B is the Rice Leaf sample in embodiment 1; C
1～C
3it is the petal sample of rape flower in embodiment 6; D
1～D
4it is the sample that in embodiment 6, rape is spent after blooming; 1 is 28-norbrassinolide, and 2 is 28-norcastasterone, and 3 is brassinolide, and 5 is castasterone.
Embodiment
Embodiment 1
The mensuration of rape element sterol in Rice Leaf
Accurately weigh 1g rice leaf and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinosteroid (brassinolide) and
2h
3the each 4ng of rape element sterone (castasterone) extracts with 12h in the refrigerator of-18 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 9 disodium hydrogen phosphate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.As shown in Figure 2, B is endogenous rape element sterol in the paddy rice detecting.For realizing the qualitative of the element of rape in sample sterol, as shown in Figure 1, the invention provides the multiple-reaction monitoring figure of the standard model of 7 kinds of rapes element sterols.The multiple-reaction monitoring pattern quantitative and qualitative analysis ion pair of 7 kinds of rape element sterols is as shown in table 1:
The multiple-reaction monitoring pattern quantitative and qualitative analysis ion pair of table 1.7 kind of rape element sterol
The rape element sterol of applying three kinds of concentration of Rice Leaf extract provided by the present invention add target relative standard deviation and the recovery as shown in table 2:
The rape element sterol of three kinds of concentration of table 2. Rice Leaf extract adds target relative standard deviation and the recovery
Can find out, the accuracy of quantitative detecting method of the present invention is high, good stability.
Embodiment 2
The mensuration of rape element sterol in rice root
Accurately weigh 1g rice root and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinolide and
2h
3the each 4ng of castasterone extracts with 12h in the refrigerator of-20 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 2 disodium hydrogen phosphate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.Embodiment 3
The mensuration of rape element sterol in paddy rice fringe
Accurately weigh 1g paddy rice fringe and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinolide and
2h
3the each 4ng of castasterone extracts with 12h in the refrigerator of-20 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 5 disodium hydrogen phosphate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.
Embodiment 4
The mensuration of rape element sterol in Culm of Rice
Accurately weigh 1g Culm of Rice and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinolide and
2h
3castasterone) each 4ng extracts with 12h in the refrigerator of-20 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 10 disodium hydrogen phosphate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.
Embodiment 5
The mensuration of rape element sterol in rape leave
Accurately weigh 1g rape leave and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinolide and
2h
3the each 4ng of castasterone extracts with 12h in the refrigerator of-20 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 9 ammonium formate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.As shown in Figure 2, A
1and A
2for the multiple-reaction monitoring figure of the endogenous rape element sterol in rape leave.Embodiment 6
The mensuration of rape element sterol in rape flower
Accurately weigh 0.5g rape flower and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinolide and
2h
3the each 4ng of castasterone extracts with 12h in the refrigerator of-20 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 9 ammonium formate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.As. as shown in Fig. 2, C
1～C
3and D
1～D
4be respectively the multiple-reaction monitoring figure of endogenous rape element sterol in rape flower and petal.
Embodiment 7
The mensuration of rape element sterol in Arabidopsis leaf
Accurately weigh 1g Arabidopsis leaf and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinolide and
2h
3the each 4ng of castasterone extracts with 12h in the refrigerator of-20 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 9 ammonium formate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.
Embodiment 8
The mensuration of rape element sterol in thaliana flower
Accurately weigh 1g thaliana flower and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinolide and
2h
3the each 4ng of castasterone extracts with 12h in the refrigerator of-20 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 9 ammonium formate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.
Embodiment 9
The mensuration of rape element sterol in tobacco leaf
Accurately weigh 1g tobacco leaf and be placed in mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in 10mL centrifuge tube, add Isotopic Internal Standard
2h
3brassinolide and
2h
3the each 4ng of castasterone extracts with 12h in the refrigerator of-20 DEG C of 5mL acetonitrile placements simultaneously.Sample, with the centrifugal 10min of rotating speed of 10000rpm, is got supernatant.In supernatant, add 250mg sodium chloride, with vortex mixed instrument concuss, 1min makes water and organic phase layering, abandons water.In centrifuge tube, add 1g anhydrous magnesium sulfate further to dewater again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then gained supernatant is crossed to double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, then with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.The residue drying up with 4mL ether dissolution, vortex extracting impurities, abandons water after leaving standstill; Adding 1mL pH is 9 ammonium formate buffer solution again, and vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get ether phase, dry up.Be settled to 100uL, enter high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, sampling volume is 30 μ L.
Claims (4)
1. a quantitative detecting method for endogenous rape element sterol in plant sample, is characterized in that, comprises the following steps:
1) extract endogenous rape element sterol: after accurately weighing plant sample, be placed in mortar, liquid nitrogen frozen be ground to Powdered after, add Isotopic Internal Standard
2h
3brassinosteroid and
2h
3rape element sterone, after extracting with acetonitrile, more than-18 DEG C of following placement 12h;
2) dewater: to step 1) extract after sample dewater;
3) Solid-Phase Extraction removal of impurities: by step 2) sample solution after the dewatering double-deck solid phase extraction column after by activation, after desorb, collect loading efflux and stripping liquid, remove the solvent in sample;
4) liquid-liquid extraction removal of impurities: by step 3) sample of gained is with ether dissolution, and vortex is abandoned water after leaving standstill, and adding pH is 2～10 buffer solution, and vortex is abandoned water after leaving standstill, and repeats removal of impurities more than 3 times or 3 times, gets ether phase, dries up constant volume;
5) constant volume detects: by step 4) sample after constant volume enters high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, measures rape element sterol.
2. the quantitative detecting method of endogenous rape element sterol in a kind of plant sample according to claim 1, is characterized in that: the filler of described double-deck solid phase extraction column is ketjenblack EC/ethylenediamine-N-propyl group bonded silica gel.
3. the quantitative detecting method of endogenous rape element sterol in a kind of plant sample according to claim 1 and 2, it is characterized in that: described dewatering is by step 1) extract after sample centrifugal, get supernatant, in supernatant, add sodium chloride, make water and organic phase layering with vortex mixed instrument concuss, abandon water, then add anhydrous magnesium sulfate to dewater in organic phase.
4. the quantitative detecting method of endogenous rape element sterol in a kind of plant sample according to claim 1 and 2, is characterized in that: described buffer solution is the aqueous solution with sodium hydrogen phosphate, the preparation of ammonium formate buffer salt.
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| CN103207103B (en) * | 2013-04-16 | 2015-11-04 | 武汉大学 | The sample-pretreating method of endogenous rape element sterol in a kind of plant sample |
| CN103558175A (en) * | 2013-10-31 | 2014-02-05 | 大连大公环境检测有限公司 | Method for determining oil substances in water sample |
| CN103713072B (en) * | 2014-01-06 | 2015-07-29 | 中国烟草总公司郑州烟草研究院 | A kind of GC-MS targeting tobacco sample sterol extracting method |
| CN104267123A (en) * | 2014-09-30 | 2015-01-07 | 中国农业科学院油料作物研究所 | Analysis method of free sterol components in edible oil sample and swill-cooked dirty oil identification method |
| CN105116060A (en) * | 2015-06-25 | 2015-12-02 | 浙江万里学院 | Rapid and efficient detection method of brassinosteroid |
| 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 |
| CN107543746A (en) * | 2016-11-16 | 2018-01-05 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and quantitative detecting method of the endogenous plant hormone of three class difference chemical property in a kind of plant sample |
| CN109813824B (en) * | 2017-11-22 | 2021-11-26 | 中国科学院大连化学物理研究所 | Pretreatment method of plant sample |
| CN110376320B (en) * | 2018-04-13 | 2021-04-27 | 中国科学院大连化学物理研究所 | Method for full-automatic series solid-phase extraction of brassinosteroids in plants |
| CN109374763B (en) * | 2018-10-18 | 2020-07-28 | 中国科学院遗传与发育生物学研究所 | Quantitative detection method for content of plant endogenous plant sulfokine |
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