CN102980953A - 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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- CN102980953A CN102980953A CN2012104865073A CN201210486507A CN102980953A CN 102980953 A CN102980953 A CN 102980953A CN 2012104865073 A CN2012104865073 A CN 2012104865073A CN 201210486507 A CN201210486507 A CN 201210486507A CN 102980953 A CN102980953 A CN 102980953A
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- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title abstract description 7
- 150000001647 brassinosteroids Chemical class 0.000 title abstract description 6
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 30
- 239000012535 impurity Substances 0.000 claims abstract description 28
- 238000000622 liquid--liquid extraction Methods 0.000 claims abstract description 4
- 239000002904 solvent Substances 0.000 claims abstract description 4
- 238000000638 solvent extraction Methods 0.000 claims abstract description 4
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 40
- 239000000523 sample Substances 0.000 claims description 40
- 239000006228 supernatant Substances 0.000 claims description 40
- 229930182558 Sterol Natural products 0.000 claims description 39
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- 235000003702 sterols Nutrition 0.000 claims description 39
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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 present invention relates to the quantitative detecting method of endogenous rape element sterol in a kind of plant sample, belong to the 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 the plant intensive amount, is generally 0.01-1ng/g.Rape element sterol is being regulated and control 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 also to elongation, division and the differentiation of vegetable cell, the increase of crop yield, reproduction, aging is to the induction of Synthesis pathway, the advolution of root, the formation of pollen tube, the activation of proton pump, photosynthesis and the specificity physiological processes such as perception of antioxidant system are played an important role.In recent years, about the physiology function of rape element sterol, for example biosynthesizing of BRs, the research of degraded and metabolic pathway is subject to extensive concern.But because the extract mesostroma complexity of plant and rape element sterol content are extremely low, carrying out of these researchs is subject to certain limitation.Therefore, set up efficient sample-pretreating method and be the key that BRs detects in conjunction with highly sensitive detection method.
At present, 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. in the plant of having reported.These methods all propose very high requirement to sample size, and majority requires more than the 40g fresh weight, and obviously so large sample size can't realize for the precious sample of part, also bring the large and high in cost of production problem of solvent consumption 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 solves the problems of the technologies described above the technical scheme that proposes to be: the quantitative detecting method of endogenous rape element sterol in a kind of plant sample may further comprise the steps:
1) extract endogenous rape element sterol: accurately be placed in the mortar behind the weighing plant sample, liquid nitrogen frozen be ground to Powdered after, add Isotopic Internal Standard [
2H
3] brassinosteroid and [
2H
3] rape element sterone, after the acetonitrile extraction, place more than the 12h below-18 ℃;
2) dewater: the sample after step 1) is extracted dewaters;
3) Solid-Phase Extraction removal of impurities: with step 2) the double-deck solid phase extraction column of the sample solution after dewatering after by activation collected loading efflux and stripping liquid after the desorb, removes the solvent in the sample;
4) liquid-liquid extraction removal of impurities: the sample of step 3) gained with ether dissolution, is abandoned water after vortex leaves standstill, add pH and be 2 ~ 10 buffer solution, abandon water after vortex leaves standstill, repeat removal of impurities more than 3 times or 3 times, get the ether phase, dry up constant volume;
5) constant volume detects: the sample behind the step 4) constant volume is entered high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, measure rape element sterol.
The filler of above-mentioned double-deck solid phase extraction column is ketjenblack EC/ethylenediamine-N-propyl group bonded silica gel.
Dewatering is that sample after step 1) is extracted is centrifugal, gets supernatant, adds sodium chloride in supernatant, makes water and organic phase layering with vortex mixed instrument concuss, abandons water, and the adding anhydrous magnesium sulfate dewaters in the organic phase again.
Buffer solution is the aqueous solution with the preparation of the buffer salts such as sodium hydrogen phosphate, ammonium formate.
Plant sample of the present invention comprises: tissue and the tissue extracts thereof such as the root of the plants such as paddy rice, rape, arabidopsis, tomato, tobacco leaf, stem, leaf, flower, pollen.
In the Solid-Phase Extraction step, ethylenediamine-N-propyl group bonded silica gel is the most frequently used adsorbent, and it shows good performance at aspects such as removing polar impurity (such as organic acid, polarity pigment, carbohydrate).In addition, the hydrophobic matrix in the 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 the plant extraction liquid is the π architecture compound on 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 is in conjunction with polarity and the non polar impurities that can effectively remove in the 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 that needs is little, realize simultaneously fast and effectively rape element sterol detection method, only spend the middle detection that realizes endogenous rape element sterol at 1g plant leaf blade and 0.5g.
Description of 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, and 4 is the standard model of 24-epibrassinolide, and 5 is the standard model of castasterone, 6 is the standard model of 24-epicastasterone, and 7 is the standard model of homobrassinolide.
Fig. 2 is the multiple-reaction monitoring figure that uses endogenous rape element sterol provided by the present invention, wherein A
1And A
2It is the rape leave sample among the embodiment 5; B is the Rice Leaf sample among the embodiment 1; C
1~ C
3It is the petal sample of rape flower among the embodiment 6; D
1~ D
4It is the bloom sample of rear flower of rape among the embodiment 6; 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 the Rice Leaf
Accurately weighing 1g rice leaf places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, add Isotopic Internal Standard [
2H
3] brassinosteroid (brassinolide) and [
2H
3] each 4ng of rape element sterone (castasterone), extract with 12h in the refrigerator of-18 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mL pH again and be 9 disodium hydrogen phosphate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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 that detects.For realizing the qualitative of rape element sterol in the sample, 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 using three kinds of concentration of Rice Leaf extract provided by the present invention add the 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 that the accuracy of quantitative detecting method of the present invention is high, good stability.
Embodiment 2
The mensuration of rape element sterol in the rice root
Accurately weighing 1g rice root places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, add Isotopic Internal Standard [
2H
3] brassinolide and [
2H
3] each 4ng of castasterone, extract with 12h in the refrigerator of-20 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mLpH again and be 2 disodium hydrogen phosphate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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.
The mensuration of rape element sterol in the paddy rice fringe
Accurately weighing 1g paddy rice fringe places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, the adding Isotopic Internal Standard [
2H
3] brassinolide and [
2H
3] each 4ng of castasterone, extract with 12h in the refrigerator of-20 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mLpH again and be 5 disodium hydrogen phosphate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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 the Culm of Rice
Accurately weighing 1g Culm of Rice places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, add Isotopic Internal Standard [
2H
3] brassinolide and [
2H
3] castasterone) each 4ng, extract with 12h in the refrigerator of-20 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mLpH again and be 10 disodium hydrogen phosphate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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 the rape leave
Accurately weighing 1g rape leave places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, add Isotopic Internal Standard [
2H
3] brassinolide and [
2H
3] each 4ng of castasterone, extract with 12h in the refrigerator of-20 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mLpH again and be 9 ammonium formate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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
2Multiple-reaction monitoring figure for the element of the endogenous rape in rape leave sterol.
Embodiment 6
The mensuration of rape element sterol in the rape flower
Accurately weighing 0.5g rape flower places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, add Isotopic Internal Standard [
2H
3] brassinolide and [
2H
3] each 4ng of castasterone, extract with 12h in the refrigerator of-20 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mL pH again and be 9 ammonium formate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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, C
1~ C
3And D
1~ D
4Be respectively the multiple-reaction monitoring figure of endogenous rape element sterol in rape flower and the petal.
Embodiment 7
The mensuration of rape element sterol in the Arabidopsis leaf
Accurately weighing 1g Arabidopsis leaf places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, add Isotopic Internal Standard [
2H
3] brassinolide and [
2H
3] each 4ng of castasterone, extract with 12h in the refrigerator of-20 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mLpH again and be 9 ammonium formate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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 the thaliana flower
Accurately weighing 1g thaliana flower places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, add Isotopic Internal Standard [
2H
3] brassinolide and [
2H
3] each 4ng of castasterone, extract with 12h in the refrigerator of-20 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mLpH again and be 9 ammonium formate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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 the tobacco leaf
Accurately weighing 1g tobacco leaf places mortar, liquid nitrogen frozen be ground to Powdered after, be transferred in the 10mL centrifuge tube, add Isotopic Internal Standard [
2H
3] brassinolide and [
2H
3] each 4ng of castasterone, extract with 12h in the refrigerator of-20 ℃ of 5mL acetonitrile placements simultaneously.Sample is got supernatant with the centrifugal 10min of the rotating speed of 10000rpm.Add 250mg sodium chloride in supernatant, 1min makes water and organic phase layering with vortex mixed instrument concuss, abandons water.Adding the 1g anhydrous magnesium sulfate in the centrifuge tube further dewaters again.With the centrifugal 10min of 10000rpm rotating speed, get supernatant.Then the gained supernatant is crossed double-deck solid phase extraction column (in advance through the activation of 5mL acetonitrile) by solid phase extraction column, again with acetonitrile/methanol (1/1, v/v, 2mL) desorb.Collect loading efflux and stripping liquid, nitrogen dries up.With the residue that the 4mL ether dissolution dries up, the vortex extracting impurities is abandoned water after leaving standstill; Add 1mL pH again and be 9 ammonium formate buffer solution, the vortex extracting impurities is abandoned water after leaving standstill; Repeat so altogether 3 times.Get the 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. the quantitative detecting method of endogenous rape element sterol in the plant sample is characterized in that, may further comprise the steps:
1) extract endogenous rape element sterol: accurately be placed in the mortar behind the weighing plant sample, liquid nitrogen frozen be ground to Powdered after, add Isotopic Internal Standard [
2H
3] brassinosteroid and [
2H
3] rape element sterone, after the acetonitrile extraction, place more than 12 h below-18 ℃;
2) dewater: the sample after step 1) is extracted dewaters;
3) Solid-Phase Extraction removal of impurities: with step 2) the double-deck solid phase extraction column of the sample solution after dewatering after by activation collected loading efflux and stripping liquid after the desorb, removes the solvent in the sample;
4) liquid-liquid extraction removal of impurities: the sample of step 3) gained with ether dissolution, is abandoned water after vortex leaves standstill, add pH and be 2 ~ 10 buffer solution, abandon water after vortex leaves standstill, repeat removal of impurities more than 3 times or 3 times, get the ether phase, dry up constant volume;
5) constant volume detects: the sample behind the step 4) constant volume is entered high performance liquid chromatography-electron spray-Tandem Mass Spectrometry Analysis, measure rape element sterol.
2. the quantitative detecting method of endogenous rape element sterol in a kind of plant sample according to claim 1, it 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 that sample after step 1) is extracted is centrifugal, get supernatant, in supernatant, add sodium chloride, make water and organic phase layering with vortex mixed instrument concuss, abandon water, add again anhydrous magnesium sulfate in the organic phase and dewater.
4. 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 buffer solution is the aqueous solution with the preparation of the buffer salts such as sodium hydrogen phosphate, ammonium formate.
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| CN103207103A (en) * | 2013-04-16 | 2013-07-17 | 武汉大学 | Sample pretreatment method of endogenous brassinosteroids in 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 |
| CN103713072A (en) * | 2014-01-06 | 2014-04-09 | 中国烟草总公司郑州烟草研究院 | GC-MS (gas chromatography-mass spectrometer) targeted tobacco sample sterol extraction 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 |
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| CN103207103A (en) * | 2013-04-16 | 2013-07-17 | 武汉大学 | Sample pretreatment method of endogenous brassinosteroids in plant sample |
| CN103207103B (en) * | 2013-04-16 | 2015-11-04 | 武汉大学 | A sample pretreatment method for endogenous brassinosterol in plant samples |
| 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 |
| CN103713072A (en) * | 2014-01-06 | 2014-04-09 | 中国烟草总公司郑州烟草研究院 | GC-MS (gas chromatography-mass spectrometer) targeted tobacco sample sterol extraction 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 |
| CN109813824A (en) * | 2017-11-22 | 2019-05-28 | 中国科学院大连化学物理研究所 | A kind of plant sample pretreatment method |
| 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 |
| CN110376320A (en) * | 2018-04-13 | 2019-10-25 | 中国科学院大连化学物理研究所 | A kind of method of rape element sterol in fully-automatic series Solid Phase Extraction plant |
| 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 |
| CN109374763A (en) * | 2018-10-18 | 2019-02-22 | 中国科学院遗传与发育生物学研究所 | Quantitative detection method of plant endogenous phytosulfopeptides |
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