CN110389178A - A method of 4 kinds of endogenous hormones in jujube are measured simultaneously using high performance liquid chromatography - Google Patents

A method of 4 kinds of endogenous hormones in jujube are measured simultaneously using high performance liquid chromatography Download PDF

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CN110389178A
CN110389178A CN201810349391.6A CN201810349391A CN110389178A CN 110389178 A CN110389178 A CN 110389178A CN 201810349391 A CN201810349391 A CN 201810349391A CN 110389178 A CN110389178 A CN 110389178A
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centrifuge tube
extraction
methanol
phase
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刘孟军
刘平
刘警
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Hebei Agricultural University
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Hebei Agricultural University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components

Abstract

The present invention provides a kind of methods for detecting 4 kinds of endogenous hormones in jujube simultaneously using high performance liquid chromatography, the analysis determination step of extraction purification step, the preparation steps of standard solution and high performance liquid chromatography including plant hormone to sample and mark product, in the extraction purification step of plant hormone, combined using cryogrinding, low-temperature dark extraction with ultrasonic wave extraction, PVPP is except impurity, ethyl acetate and extracting n-butyl alcohol, C18The modes such as Solid Phase Extraction column separating purification, it is ensured that the abundant extraction of plant hormone.Method of the invention has many advantages, such as that high sensitivity, sample extraction are relatively simple, the rate of recovery is high and accuracy and precision are high, the detection of endogenous hormones suitable for jujube multiclass tissue.

Description

A method of 4 kinds of endogenous hormones in jujube are measured simultaneously using high performance liquid chromatography
Technical field
The present invention relates to technical field of chromatographic analysis, and in particular to a kind of to be measured 4 in jujube simultaneously using high performance liquid chromatography The method of kind endogenous hormones.
Technical background
Jujube tree (Ziziphus jujubaMill.) for Rhamnaceae (Rhamnaceae) zizyphus (ZiziphusMill. it) plants Object is the big dry fruit tree variety in China first and China's characteristic and advantage fruit tree.China's jujube cultivation gross area about 2,000,000 at present hm2, close to apple and citrus, it is the first that yield is sure to occupy China's dry fruit.Plant endogenous hormones are small point of the one kind generated in plant Sub- compound can play a variety of physiological actions, each process of regulating growth of plants under extremely low concentration.But it is endogenous Hormone is plant in-vivo content is extremely low, property is unstable, therefore, plant endogenous to swash vulnerable to the interference of other compounds in cell The measuring method of element must be very sensitive and single-minded.Traditional measuring method is surveyed while being difficult to realize plant a variety of endogenous hormones It is fixed.
Plant hormone is many kinds of, and the six major class plant endogenous hormones generally acknowledged at present by scientific circles have plant auxins (auxins), gibberellin class (gibberenllins, GA), cytokinin (cytokinins, CK), ethylene (ethylene), abscisic acid (abscisic acid, ABA), brassinosteroid (Brassinosteroids, BRs).No Same hormone kind plays different physiological actions.In the growth and development process of plant, each parahormone plays difference respectively Effect, and Dressing date and concentration can all influence the developmental condition of plant, since plant has the uniqueness of micro stimulation Feature, thus research report at home and abroad is more.
Plant endogenous hormones participate in each stage of development of plant, adjust the vital movement of plant.Therefore, it is carried out Quickly accurate measurement is very important.The measuring method of plant hormone has very much, can be divided into biology according to its measuring principle Identification method, Physico-chemical tests method and immunodetection.Wherein, high performance liquid chromatography is current more reliable detection method, tool Have the advantages that sensitivity and selectivity are high, reproducible, but the report for measuring on plant various plants hormone simultaneously is less, and Variety classes plant hormone has synergistic effect in the various physiology courses of regulation plant, therefore, utilizes high performance liquid chromatography Accurate Determining multiclass plant hormone is still a problem simultaneously.The present invention passes through optimization endogenous hormones extracting method and chromatographic condition, A kind of quick, reliable method is provided to measure 4 kinds of endogenous hormones in jujube simultaneously.
Summary of the invention
The present invention provides a kind of methods for measuring 4 kinds of endogenous hormones in jujube simultaneously using high performance liquid chromatography, have spirit The advantages that sensitivity is high, sample extraction is relatively simple, the rate of recovery is high and accuracy and precision are high, the analysis that can satisfy trace is wanted It asks.
A method of 4 kinds of endogenous hormones in jujube are measured simultaneously using high performance liquid chromatography, comprising:
(1) jujube tissue 1g is taken, and 80% 6~10ml of methanol that pre-cooling is added is ground to homogenate, is transferred in the first centrifuge tube, low-temperature dark Extract 12~15h；
(2) ultrasonic wave extraction 15min；
(3) 4 DEG C of 13000rmin of extracting solution-1It is centrifuged 15min, supernatant is transferred in the second centrifuge tube, is dried with nitrogen to water Phase；
(4) by aforementioned water phase 1molL-1NaOH adjusts pH to 8.0, and PVPP, 4 DEG C of 10000r after sonic oscillation is added min-1It is centrifuged 10min, supernatant is transferred in third centrifuge tube, 1molL is used-1Lemon acid for adjusting pH the bodies such as is added to 3.0 Long-pending ethyl acetate extraction, repetitive operation this step 1~3 time, ethyl acetate is sucked out in the 4th centrifuge tube, water phase is stand-by；
(5) by aforementioned water phase 1molL-1NaOH adjusts pH to 8.0, and isometric extracting n-butyl alcohol, repetitive operation sheet is added N-butanol is sucked out in the 5th centrifuge tube for step 1~3 time；
(6) aforementioned ester is mutually dried with nitrogen, the acetic acid of 2ml pH 3.5 is added, obtains crude extract 1；
(7) aforementioned alcohol phase is dried with nitrogen, the ammonium acetate dissolution of 2ml pH 7.5 is added, obtains crude extract 2；
(8) C is used18Solid-phase extraction column isolates and purifies crude extract 1 and 2 respectively, merges, and crosses miillpore filter and obtains to test sample Product；
(9) sample to be tested is analyzed using high performance liquid chromatography, chromatographic condition are as follows: chromatographic column: Agilent ZORBAX ODS-C18(4.6 × 150mm, 5 μm)；0.6% glacial acetic acid of mobile phase: Yi Jing ﹕ Jia Chun ﹕=5 ﹕, 55 ﹕ 40；Sample volume: 10 μ L；Column temperature: 30℃；Detection wavelength: 254nm；It is quantitative determined using external standard method, obtains 4 kinds of endogenous hormones i.e. zeatin in jujube tissue, red The content of mycin, auxin and abscisic acid.
The method for measuring 4 kinds of endogenous hormones in jujube simultaneously according to the present invention, steps are as follows:
It accurately weighs 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration that 8ml pre-cooling is added is transferred at homogenate In first centrifuge tube, 4 DEG C are protected from light 12~15h of extraction, ultrasonic oscillation 15min, 4 DEG C of 13000rmin-1It is centrifuged 15min；It receives Collect supernatant in the second centrifuge tube, is dried with nitrogen methanol, water phase 1molL-10.1g is added in NaOH tune pH to 8.0 PVPP, ultrasonic 30min, 10000rmin-1It is centrifuged 10min；Supernatant is collected in third centrifuge tube, uses 1molL-1Lemon Lemon acid tune pH to 3.0 is added isometric ethyl acetate and extracts 3 times, merges ester phase in the 4th centrifuge tube；Water phase 1molL-1 NaOH tune pH to 8.0, is added isometric extracting n-butyl alcohol 3 times, merges alcohol phase in the 5th centrifuge tube；It is dried with nitrogen ester phase, is added The acetic acid for entering 2ml pH 3.5 is dried with nitrogen alcohol phase, and the ammonium acetate dissolution of 2ml pH 7.5 is added, crosses C respectively18Solid phase extraction Column is taken, the elution of 10% methanol is eluted with 1ml alkaline methanol and acidic methanol respectively, merges eluent, crosses 0.45 μm of miillpore filter Sample to be tested is obtained, it is to be measured to be placed in 4 DEG C of refrigerators preservations；Sample to be tested is analyzed using high performance liquid chromatography, chromatographic condition Are as follows: chromatographic column: Agilent ZORBAX ODS-C18(4.6 × 150mm, 5 μm)；0.6% glacial acetic acid of mobile phase: Yi Jing ﹕ Jia Chun ﹕= 5 ﹕, 55 ﹕ 40；Sample volume: 10 μ L；Column temperature: 30 DEG C；Detection wavelength: 254nm；It is quantitative determined using external standard method, obtains jujube group The 4 kinds of endogenous hormones i.e. content of zeatin, gibberellin, auxin and abscisic acid in knitting.
Compared with prior art, it is had the advantages that in above-mentioned technical proposal
1. inventor has found in completing process of the present invention, ice bath grinding is not able to maintain vegetable material and has been at a low temperature In environment.Mortar is placed in -20 DEG C of refrigerators before the milling and is pre-chilled, it is ensured that vegetable material is during the grinding process always in low In warm environment.
2. the present invention removes organic phase methanol, ethyl acetate and n-butanol using nitrogen evaporator, easy to operate, rapidly and efficiently, The degradation of hormone can be reduced to a certain extent.
3. the present invention removes depigmentaton and phenolic substances using PVPP, the interference of impurity can be efficiently reduced, makes to extract process It is simplified, reduces the loss of plant hormone.
4. the present invention uses C18Solid-phase extraction column purifies plant hormone crude extract, can effectively remove impurity, and It has no adverse effects to endogenous hormones.
5. the present invention uses the design of isocratic elution, it is effectively saved the analysis time of sample, rapidly and efficiently.
6. the selection of flow velocity can guarantee that chromatographic peak peak shape is best, flow velocity is too fast to will appear overlap peak, and flow velocity can make peak slowly excessively Deformation is wide, when flow velocity is 0.8mlmin-1When each hormone peak shape it is best.
Detailed description of the invention
Fig. 1 is the chromatographic determination result of standard items of the invention；
Fig. 2 is the chromatographic determination result of sample liquid of the invention；
Fig. 3 is the chromatographic determination result of extracting method (3)；
Fig. 4 is that mobile phase is 55% methanol: the chromatographic determination result of 45% water (glacial acetic acid containing 0.6%)；
1 is GA in figure3, 2 be IAA, and 3 be ABA, and 4 be ZT.
Specific embodiment
Embodiment 1
The present embodiment mainly uses following instrument and reagent:
Instrument: 1200 high performance liquid chromatograph of U.S. Agilent (including on-line degassing machine, quaternary pump, autosampler, column temperature Case, UV detector, chromatographic work station)；Ohaus Instrument's acidometer；KQ-500DE type numerical control supersonic Washer (Kunshan Ultrasonic Instruments Co., Ltd.)；(Jintan City's Jie Ruier electric appliance is limited for TGL-16 high speed desktop electric centrifuge Company)；BS124S type electronic balance (Sai Duolisi instrument system Co., Ltd, Beijing)；III circulating water type of SHD- is mostly used vacuum It pumps (Baoding high and new technology industrial development zone sunlight Science & Teaching Instrument factory)；2000D type Superpure water machine (mayor of Beijing bearing instruments and meters company)；N-EVAP 112 nitrogen evaporators (U.S. Organomation).
Reagent: acetonitrile, methanol (chromatographically pure, Tianjin good fortune morning chemical reagent factory)；(the Beijing polyvinylpyrrolidone (PVPP) Solarbio)；Ethyl acetate (Tianjin Fu Yu Fine Chemical Co., Ltd)；(Tianjin is into the limited public affairs of Feng Huagong for n-butanol Department)；Citric acid, NaOH(good fortune morning chemistry)；Ultrapure water；GA3, IAA, ABA and ZT standard items (HPLC grades, purity >=98%, Beijing Solarbio production).
The method that the present invention is while measuring 4 kinds of endogenous hormones in jujube, can be used for measuring jujube tissue (branch skin, callus group Knit, pericarp, pulp, seed etc.) in GA3, IAA, ABA and ZT content.
The method that the present embodiment measures 4 kinds of endogenous hormones in jujube simultaneously, the system of extraction, standard solution including endogenous hormones Standby and efficient liquid phase chromatographic analysis determination step.
1. the extraction step of plant hormone
It accurately weighs jujube callus 1.0000g to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, ultrasonic oscillation 15min, 4 DEG C of 13000rmin-1Centrifugation 15min collects supernatant in the second centrifuge tube, is dried with nitrogen methanol, water phase 1molL-1NaOH tune pH to 8.0 is added 0.1g PVPP, ultrasonic 30min, 10000rmin-1It is centrifuged 10min, supernatant is collected in third centrifuge tube, uses 1mol L-1Citric acid tune pH to 3.0 is added isometric ethyl acetate and extracts 3 times, merges ester mutually in the 4th centrifuge tube, water phase is used 1mol·L-1NaOH tune pH to 8.0, is added isometric extracting n-butyl alcohol 3 times, merges alcohol phase in the 5th centrifuge tube, nitrogen is blown The acetic acid of 2ml pH 3.5 is added in dry ester phase, is dried with nitrogen alcohol phase, the ammonium acetate dissolution of 2ml pH 7.5 is added, respectively Cross C18Solid-phase extraction column, 10% methanol elution, is eluted with 1ml alkaline methanol and acidic methanol respectively, merges eluent, crosses 0.45 μ M miillpore filter obtains sample to be tested, and it is to be measured to be placed in 4 DEG C of refrigerators preservations.
Above-mentioned first, second, third, fourth, the 5th centrifuge tube only has the function of distinguishing object do not have practical meaning Justice.In the present embodiment, the extracting method of endogenous hormones is to have done a large amount of exploration and research on the basis of existing technology, and most Embodiment after being optimized eventually.Inventor tests the extracting method of following 12 kinds of endogenous hormones respectively:
(1) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration that 8ml pre-cooling is added turns at homogenate Enter in the first centrifuge tube, 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in the second centrifugation Guan Zhong is dried with nitrogen methanol, isometric petroleum ether extraction is added 3 times, discards petroleum ether, water phase Na2HPO4PH to 8.0 is adjusted, 0.02g PVPP ultrasonic vibration 30min, 7500rmin is added-1It is centrifuged 10min, supernatant is collected in third centrifuge tube, uses 1mol·L-1 Lemon acid for adjusting pH is added isometric ethyl acetate and extracts 3 times to 3.0, merges ester mutually in the 4th centrifuge tube, It is dried with nitrogen ester phase, it is to be measured to cross 0.45 μm of miillpore filter for the dissolution of 1ml methanol after concussion.Water phase 1molL-1 NaOH is adjusted PH to 8.0, equivalent are added extracting n-butyl alcohol 3 times, merge alcohol phase in the 5th centrifuge tube, are dried with nitrogen alcohol phase, 1ml methanol is molten It is to be measured to cross 0.45 μm of miillpore filter for solution after concussion.
(2) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light extraction for 24 hours, 10000rmin-1First centrifugation 15min, collects supernatant in second In centrifuge tube, peracidity methanol activates the C of 12h18Solid-phase extraction column, acidic methanol elution collect leacheate and (include alkaline hormone ZT) in third centrifuge tube, alkaline methanol elution collects eluent and (includes acid hormone GA3, IAA, ABA) in the 4th centrifugation It is to be measured that 0.45 μm of miillpore filter is crossed in pipe.By leacheate 1molL in third centrifuge tube-1 NaOH tune pH to 8.0, parlkaline The C of methanol activation 12h18Solid-phase extraction column, alkaline methanol elution, acidic methanol elution collect eluent (including ZT) in the 5th In centrifuge tube, it is to be measured to cross 0.45 μm of miillpore filter.
(3) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, residue is added 80% methanol of 5ml and repeats to extract 6h, 10000rmin-1It is centrifuged 15min, merges supernatant, is added 0.2g PVPP, ultrasonic vibration 30min, 10000rmin-1Third is centrifuged 10min, collects supernatant in third centrifuge tube, Cross C18Solid-phase extraction column collects efflux in the 4th centrifuge tube, and the dissolution of 2ml methanol, 10000r is added in freeze-drying min-14th centrifugation 3min, it is (chromatographic determination result is shown in Fig. 3) to be measured that supernatant crosses 0.45 μm of miillpore filter.
(4) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, it is dried with nitrogen methanol, isometric petroleum ether extraction is added 3 times, ether phase is discarded, uses 1molL-1 NaOH tune pH is extremely 8.0,0.1g PVPP, ultrasonic vibration 30min, 10000rmin is added-1It is centrifuged 10min, collects supernatant in third centrifuge tube In, use 1molL-1 Citric acid tune pH to 3.0 is added isometric ethyl acetate and extracts 3 times, merges ester mutually in the 4th centrifuge tube In, it is dried with nitrogen ester phase, the acetic acid of 2ml pH 3.5 is added, crosses C18Solid-phase extraction column, the elution of 10% methanol, 1ml alkalinity Methanol elution, it is to be measured to cross 0.45 μm of miillpore filter.
(5) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, 0.2g PVPP, ultrasonic vibration 30min, 10000rmin is added-1Be centrifuged 10min, collect supernatant in third from In heart pipe, with acetic acid tune pH to 3.0, C is crossed180.45 μm of micropore filter is crossed in solid-phase extraction column, the elution of 10% methanol, alkaline methanol elution Film is to be measured.
(6) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, it is dried with nitrogen methanol, isometric petroleum ether extraction is added 3 times in water phase, discards ether phase, and 0.2g PVPP is added, and surpasses 30min, 10000rmin are swung in acoustic shock-1It is centrifuged 10min, collects supernatant in third centrifuge tube, with acetic acid tune pH to 3.0, etc. Volume of ethylacetate extracts 3 times, merges ester mutually in the 4th centrifuge tube, water phase 1molL-1NaOH tune pH to 8.0 waits bodies Product extracting n-butyl alcohol 3 times, merges alcohol phase in the 5th centrifuge tube, be dried with nitrogen ester phase and alcohol phase, respectively plus 1ml methanol dissolves, Merge, crosses C18It is to be measured to cross 0.45 μm of miillpore filter for solid-phase extraction column, the elution of 10% methanol of 6ml, the elution of 1ml alkaline methanol.
(7) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, it is dried with nitrogen methanol, isometric petroleum ether extraction is added 2 times in water phase, stands 8h, discards ether phase, and water phase is used NaH2PO4And Na2HPO4PH to 6.4 is adjusted, 0.2g PVPP, ultrasonic vibration 30min, 10000rmin is added-1It is centrifuged 10min, is received Collect supernatant in third centrifuge tube, with acetic acid tune pH to 2.9, isometric ethyl acetate is extracted 3 times, every minor tick 8h, is merged Ester mutually in the 4th centrifuge tube, is dried with nitrogen ester phase, and 1ml methanol is added to dissolve, and it is to be measured to cross 0.45 μm of miillpore filter.
(8) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, it is dried with nitrogen methanol, isometric petroleum ether extraction is added 2 times in water phase, stands 8h, discards ether phase, and water phase is used NaH2PO4And Na2HPO4PH to 6.4 is adjusted, 0.2g PVPP, ultrasonic vibration 30min, 10000rmin is added-1It is centrifuged 10min, is received Collect supernatant in third centrifuge tube, with acetic acid tune pH to 2.9, isometric ethyl acetate is extracted 3 times, every minor tick 8h, is merged Ester mutually in the 4th centrifuge tube, is dried with nitrogen ester phase, and 3.5 acetic acid of 2ml pH is added and redissolves, crosses C18Solid-phase extraction column, 10% methanol Elution, 1ml alkaline methanol elution, it is to be measured to cross 0.45 μm of miillpore filter.
(9) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, nitrogen is blown to close dry, addition 5ml ammonium acetate solution (0.1 molL-1, pH 9) redissolve, at 4 DEG C 13000r·min-1It is centrifuged 30min, supernatant is collected in third centrifuge tube, 0.2g PVPP ultrasonic vibration 30min is added, 10000r·min-1It is centrifuged 10min, supernatant is collected in the 4th centrifuge tube, with 1 molL-1NaOH tune pH to 8.0, mistake C18Solid-phase extraction column is (with 0.1 molL-1Ammonium acetate balance), 0.01 molL-1With 0.1 molL-1Ammonium acetate elution, The elution of 1ml chromatography methanol；Sample efflux and leacheate are collected in the 5th centrifuge tube, uses 1molL-1Citric acid tune pH is extremely 3.0, it crosses with 0.1 molL-1The equilibrated C of acetic acid18Solid-phase extraction column, 0.1 molL-1With 1.5 molL-1Acetic acid leaching It washes, it is to be measured to cross 0.45 μm of miillpore filter for the elution of 1ml chromatography methanol.
(10) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, nitrogen, which is blown to, closely to be done, and uses 1molL-1Citric acid tune pH to 3.5 crosses C18Solid-phase extraction column, the elution of 10% methanol, Methanol or alkaline methanol elution, it is to be measured to cross 0.45 μm of miillpore filter.
(11) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, it is dried with nitrogen methanol, water phase is taken out and thaws in -20 DEG C of freezing 30min, 13000rmin-1It is centrifuged 10min, is received Collect supernatant in third centrifuge tube, uses 1molL-1Citric acid tune pH to 3.0 crosses C18Solid-phase extraction column (pure methanol 3ml+ 0.1mol·L-1Formic acid activation), 6ml 0.1molL-1Formic acid elution, alkaline methanol elution are crossed 0.45 μm of miillpore filter and are waited for It surveys.
(12) accurately weigh 1.0000g sample to be put into the mortar of pre-cooling, 80% methanol trituration of 8ml pre-cooling is added at even Slurry is transferred in the first centrifuge tube, and 4 DEG C are protected from light 12~15h of extraction, 10000rmin-1It is centrifuged 15min, collects supernatant in second In centrifuge tube, it is dried with nitrogen methanol, 1 molL of water phase-10.1g PVPP, ultrasonic vibration is added in NaOH tune pH to 8.0 30min, 10000rmin-1It is centrifuged 10min, supernatant is collected in third centrifuge tube, isometric extracting n-butyl alcohol is added 3 times, Merge alcohol phase in the 4th centrifuge tube, water phase 1molL-1Isometric ethyl acetate extraction 3 is added in citric acid tune pH to 3.0 It is secondary, merge ester phase in the 5th centrifuge tube, be dried with nitrogen alcohol phase, the ammonium acetate dissolution of 2ml pH 7.5 is added, is dried with nitrogen ester The acetic acid of 2ml pH 3.5 is added in phase, crosses C respectively18Solid-phase extraction column, 10% methanol elution, uses 1ml acidic methanol respectively It is eluted with alkaline methanol, it is to be measured to cross 0.45 μm of miillpore filter.
Inventors have found that plant hormone extraction step is most important.Hormone is extracted and is purified during above method test Effect is undesirable, and impurity is more and the rate of recovery is very low.Browning easily occurs for plant tissue during the grinding process, so inventor is grinding Mortar is placed in -20 DEG C of refrigerators before mill and is pre-chilled, the low temperature environment in process of lapping can be kept brown to prevent to a certain extent Change.Point out that petroleum ether can be used as the solvent of depigmentation according to existing document, but inventor has found in completing process of the present invention It goes depigmentation effect unobvious using petroleum ether and emulsion is be easy to cause to be difficult to be layered.Pass through a large number of experiments, invention human hair Pigment and phenolic substances, the layering for not influencing water phase and extractant now can be effectively removed using PVPP, and will not be to plant hormone Cause adverse effect, simple and effective and the extracting method for finally determining plant hormone are as follows: accurately weigh 1.0000g sample and be put into In the mortar of pre-cooling, be added 8ml pre-cooling 80% methanol trituration be transferred in the first centrifuge tube at homogenate, 4 DEG C be protected from light extraction 12~ 15h, ultrasonic oscillation 15min, 4 DEG C of 13000rmin-1It is centrifuged 15min, collects supernatant in the second centrifuge tube, nitrogen is blown Dry methanol, water phase 1molL-1 0.1g PVPP, ultrasonic 30min, 10000rmin is added in NaOH tune pH to 8.0-1Centrifugation 10min collects supernatant in third centrifuge tube, uses 1molL-1 Isometric ethyl acetate is added in citric acid tune pH to 3.0 Extraction 3 times merges ester mutually in the 4th centrifuge tube, water phase 1molL-1 Isometric n-butanol is added in NaOH tune pH to 8.0 Extraction 3 times merges alcohol phase in the 5th centrifuge tube, is dried with nitrogen ester phase, the acetic acid of 2ml pH 3.5 is added, is dried with nitrogen Alcohol phase is added the ammonium acetate dissolution of 2ml pH 7.5, crosses C respectively18Solid-phase extraction column, 10% methanol elution, respectively with 1ml alkalinity Methanol and acidic methanol elution, merge eluent, cross 0.45 μm of miillpore filter and obtain sample to be tested, be placed in 4 DEG C of refrigerators save to It surveys.
2. the preparation step of standard solution
Accurately weigh GA3, each 0.005g of IAA, ABA, ZT standard items, respectively with methanol constant volume to 10ml, being configured to concentration is 500mg·L-1Standard Reserving Solution, a certain amount of Standard Reserving Solution is respectively drawn with liquid-transfering gun, is diluted to 50mgL step by step-1、 20mg·L-1、5mg·L-1、2mg·L-1、0.5mg·L-1Mixed standard solution, keep in dark place at low temperature spare.
3. efficient liquid phase chromatographic analysis determination step
Chromatographic condition:
Chromatographic column: Agilent ZORBAX ODS-C18(4.6 × 150mm, 5 μm)；0.6% glacial acetic acid of mobile phase: Yi Jing ﹕ Jia Chun ﹕ =5:55:40；Sample volume: 10 μ L；Column temperature: 30 DEG C；Flow velocity: 0.8mlmin-1；Detection wavelength: 254nm；
It is quantitative determined using external standard method.
In the prior art, most common mobile phase is the combination of methanol-water.Inventor tests 35% methanol respectively: 65% Water (glacial acetic acid containing 0.6%), 40% methanol: 60% water (glacial acetic acid containing 0.6%), 45% methanol: 55% water is (containing 0.6% ice second Acid), 55% methanol: 45% water (glacial acetic acid containing 0.6%) (chromatographic determination result is shown in Fig. 4), 60% methanol: 40% water is (containing 0.6% ice Acetic acid) elution effect, discovery chromatographic peak peak shape is bad and separating degree is poor, chromatography overlap of peaks, and 4 kinds of hormones to be measured in sample are not It can efficiently separate.
It is documented, acetonitrile can improve peak shape, keep peak more sharp, and improve separating degree.Inventor tests respectively 5% acetonitrile: 50% methanol: 45% water (glacial acetic acid containing 0.6%), 5% acetonitrile: 45% methanol: 50% water (glacial acetic acid containing 0.6%), 5% Acetonitrile: 60% methanol: 35% water (glacial acetic acid containing 0.6%), 10% acetonitrile: 45% methanol: 45% water (glacial acetic acid containing 0.6%), 15% Acetonitrile: 40% methanol: 45% water (glacial acetic acid containing 0.6%), 15% acetonitrile: 35% methanol: 50% water (glacial acetic acid containing 0.6%), 5% second Nitrile: 55% methanol: the elution effect of 40% water (glacial acetic acid containing 0.6%), discovery use 5% acetonitrile: 55% methanol: 40% water (contains 0.6% glacial acetic acid) it is used as mobile phase that each hormone can be made to efficiently separate, and peak shape is fine.
The change of flow rate of mobile phase will affect the retention time of hormone.Flow velocity is too fast to will appear overlap peak, and flow velocity slowly can excessively Peak shape is set to broaden.Experimental Comparison 0.4mlmin-1、0.6ml·min-1、0.8ml·min-1、1.0ml·min-1With 1.2ml·min-1This 5 kinds of flow velocitys, discovery are 0.8mlmin when flow velocity-1When each hormone peak shape it is best.
The standard working curve and detection limit of 1 endogenous hormones of table
Endogenous hormones Regression equation Related coefficient The range of linearity (mgL-1) Detection limit LOD(mgL-1)
GA3 y=79.938x 0.9954 0.058-1.118 0.010
IAA y=26.924x 0.9913 0.045-2.133 0.001
ABA y=61.547x 0.9987 0.029-0.316 0.001
ZT y=56.319x 0.9948 0.074-1.998 0.010
As it can be seen from table 1 each concentration of standard solution and its peak area linear relationship are good, meet analysis and require.
Sample hormone-content measurement: the sample prepare liquid that step 1 is extracted filtered under determining chromatographic condition sample introduction into The peak area measured is substituted into GA in regression equation calculation jujube sample by row measurement3, IAA, ABA, ZT content.
Recovery test: drawing a certain amount of standard solution, carries out by the method for step 1 is synchronous with sample, through efficient liquid Phase chromatographic determination obtains peak area, calculates the rate of recovery.
2 endogenous hormones rate of recovery experimental result of table
Endogenous hormones Measured value (mgL-1) Measured amount (mgL-1) The rate of recovery (%)
GA3 1.32 1.11 84.09
IAA 23.56 19.48 82.68
ABA 11.93 9.25 77.54
ZT 9.98 8.25 82.67
As can be seen from Table 2, for the rate of recovery between 77%~85%, average recovery rate 81.75% meets hormone determination requirement.

Claims (1)

1. a kind of method for measuring 4 kinds of endogenous hormones in jujube simultaneously using high performance liquid chromatography, including the following steps:
(1) jujube tissue 1g is taken, and 80% 6~10ml of methanol that pre-cooling is added is ground to homogenate, is transferred in the first centrifuge tube, low-temperature dark Extract 12~15h；
(2) ultrasonic wave extraction 15min；
(3) 4 DEG C of 13000r/min of extracting solution are centrifuged 15min, and supernatant is transferred in the second centrifuge tube, is dried with nitrogen to water phase；
(4) aforementioned water phase 1M NaOH is adjusted into pH to 8.0, PVPP, 4 DEG C of 10000r/min centrifugations after sonic oscillation are added Supernatant is transferred in third centrifuge tube by 10min, and with 1M lemon acid for adjusting pH to 3.0, isometric ethyl acetate extraction is added It takes, repetitive operation this step 1~3 time, ethyl acetate is sucked out in the 4th centrifuge tube, water phase is stand-by；
(5) aforementioned water phase NaOH is adjusted into pH to 8.0, be added isometric extracting n-butyl alcohol, repetitive operation this step 1~3 It is secondary, n-butanol is sucked out in the 5th centrifuge tube；
(6) aforementioned ester is mutually dried with nitrogen, the acetic acid of 2ml pH 3.5 is added, obtains crude extract 1；
(7) aforementioned alcohol phase is dried with nitrogen, the ammonium acetate dissolution of 2ml pH 7.5 is added, obtains crude extract 2；
(8) C is used18Solid-phase extraction column isolates and purifies crude extract 1 and 2 respectively, merges, and crosses miillpore filter and obtains to test sample Product；
(9) sample to be tested is analyzed using high performance liquid chromatography, chromatographic condition are as follows: chromatographic column: Agilent ZORBAX ODS-C18(4.6 × 150mm, 5 μm)；0.6% glacial acetic acid of mobile phase: Yi Jing ﹕ Jia Chun ﹕=5 ﹕, 55 ﹕ 40；Sample volume: 10 μ L；Column temperature: 30℃；Detection wavelength: 254nm；It is quantitative determined using external standard method, obtains 4 kinds of endogenous hormones i.e. zeatin in jujube tissue, red The content of mycin, auxin and abscisic acid.
CN201810349391.6A 2018-04-18 2018-04-18 A method of 4 kinds of endogenous hormones in jujube are measured simultaneously using high performance liquid chromatography Pending CN110389178A (en)

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