CN109239226B - A method of it improves while 10 kinds of gibberellin of detection detects stability - Google Patents
A method of it improves while 10 kinds of gibberellin of detection detects stability Download PDFInfo
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- CN109239226B CN109239226B CN201811230372.8A CN201811230372A CN109239226B CN 109239226 B CN109239226 B CN 109239226B CN 201811230372 A CN201811230372 A CN 201811230372A CN 109239226 B CN109239226 B CN 109239226B
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- 238000001514 detection method Methods 0.000 title claims abstract description 73
- 229930191978 Gibberellin Natural products 0.000 title claims abstract description 53
- 239000003448 gibberellin Substances 0.000 title claims abstract description 53
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 46
- 238000003908 quality control method Methods 0.000 claims abstract description 43
- 238000000605 extraction Methods 0.000 claims abstract description 37
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 24
- CVZZNRXMDCOHBG-UHFFFAOYSA-N 2-azaniumyl-3-(2-chlorophenyl)propanoate Chemical compound OC(=O)C(N)CC1=CC=CC=C1Cl CVZZNRXMDCOHBG-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 14
- 238000002604 ultrasonography Methods 0.000 claims abstract description 14
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000019253 formic acid Nutrition 0.000 claims abstract description 12
- 238000012360 testing method Methods 0.000 claims abstract description 10
- 238000000227 grinding Methods 0.000 claims abstract description 5
- 239000000047 product Substances 0.000 claims description 45
- 230000014759 maintenance of location Effects 0.000 claims description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 14
- ICSNLGPSRYBMBD-UHFFFAOYSA-N 2-aminopyridine Chemical compound NC1=CC=CC=N1 ICSNLGPSRYBMBD-UHFFFAOYSA-N 0.000 claims description 12
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Chemical compound CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 10
- ZTNANFDSJRRZRJ-UHFFFAOYSA-N 2,4-dimethylquinoline Chemical compound C1=CC=CC2=NC(C)=CC(C)=C21 ZTNANFDSJRRZRJ-UHFFFAOYSA-N 0.000 claims description 9
- 150000002500 ions Chemical class 0.000 claims description 8
- 239000004471 Glycine Substances 0.000 claims description 7
- AKCRQHGQIJBRMN-UHFFFAOYSA-N 2-chloroaniline Chemical compound NC1=CC=CC=C1Cl AKCRQHGQIJBRMN-UHFFFAOYSA-N 0.000 claims description 6
- PNPCRKVUWYDDST-UHFFFAOYSA-N 3-chloroaniline Chemical compound NC1=CC=CC(Cl)=C1 PNPCRKVUWYDDST-UHFFFAOYSA-N 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- PJSFRIWCGOHTNF-UHFFFAOYSA-N Sulphormetoxin Chemical compound COC1=NC=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=C1OC PJSFRIWCGOHTNF-UHFFFAOYSA-N 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
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- 150000002208 flavanones Chemical class 0.000 claims description 6
- 235000011981 flavanones Nutrition 0.000 claims description 6
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 claims description 6
- 229960000282 metronidazole Drugs 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 229960004673 sulfadoxine Drugs 0.000 claims description 6
- 125000005425 toluyl group Chemical group 0.000 claims description 6
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 claims description 5
- MPDGHEJMBKOTSU-UHFFFAOYSA-N Glycyrrhetinsaeure Natural products C12C(=O)C=C3C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(C)C MPDGHEJMBKOTSU-UHFFFAOYSA-N 0.000 claims description 5
- 229960003720 enoxolone Drugs 0.000 claims description 5
- SRLROPAFMUDDRC-INIZCTEOSA-N ethyl N-benzoyl-L-tyrosinate Chemical compound C([C@@H](C(=O)OCC)NC(=O)C=1C=CC=CC=1)C1=CC=C(O)C=C1 SRLROPAFMUDDRC-INIZCTEOSA-N 0.000 claims description 5
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- 239000003960 organic solvent Substances 0.000 claims description 5
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 4
- JBFHTYHTHYHCDJ-UHFFFAOYSA-N gamma-caprolactone Chemical compound CCC1CCC(=O)O1 JBFHTYHTHYHCDJ-UHFFFAOYSA-N 0.000 claims description 4
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- 230000002159 abnormal effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
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- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
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- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
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- 238000004817 gas chromatography Methods 0.000 description 1
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
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- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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- 239000003381 stabilizer Substances 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- BAQAVOSOZGMPRM-QBMZZYIRSA-N sucralose Chemical compound O[C@@H]1[C@@H](O)[C@@H](Cl)[C@@H](CO)O[C@@H]1O[C@@]1(CCl)[C@@H](O)[C@H](O)[C@@H](CCl)O1 BAQAVOSOZGMPRM-QBMZZYIRSA-N 0.000 description 1
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Classifications
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention discloses a kind of methods of 10 kinds of gibberellin detection stability of raising while detection comprising, the extraction of 10 kinds of gibberellin: the grinding of crops blade is added and extracts reagent, ultrasound, extraction of ocean eddies；The purifying of sample；It tests and analyzes: being measured using ultra performance liquid chromatography-tandem mass spectrometer, calculate the content of gibberellin to be measured.The present invention using the 70% second eyeball containing 0.1% formic acid in pre-treatment as extraction reagent, the method that selects room temperature ultrasound 5min+ vortex 5min by adjusting extracting, select MAX and C18 (CNW LC-C18 200mg) according to mass ratio 1:(2~4) as purified material, and it uses 17 to mix and is denoted as monitoring sample acquisition situation as internal standard for general quality-control product monitoring instrument state, 2- chlorophenylalanine, substantially increase the detection stability of gibberellin of the present invention, detection repeats, and error is small.
Description
Technical field
The invention belongs to the detection technique fields of trace plant hormone, and in particular to it is a kind of improve simultaneously detect 10 kinds it is red mould
The method of element detection stability.
Background technique
Gibberellin (Gibberellins) is a kind of plant growth regulator for belonging to diterpenoids compound, is sprouted in seed
Hair, growth of seedling, yielding positive results etc. has different physiological roles, be widely used melon fruits and vegetables, cereal crops,
In the production processes such as gardening.
Since content of the gibberellin in plant is lower, the higher pre-treatment of quantitative requirement and detection method are carried out to it,
Current detection method mainly has fluorescent spectrometry, enzyme-linked immunization, gas chromatography etc., some preceding places of current detection method
Reason is excessively cumbersome, some pre-treatment sensitivity are lower, some pre-treatments will cause the pollution to instrument, detect current detection side
The most poor repeatability of method, testing result be easy operated, instrument fluctuation etc. influence so that twice repeat detection quantitative result
It differs greatly.Therefore how a kind of pollution that can reduce to instrument is provided, simplify cumbersome operating procedure bring error, increase
The stability that detects by force shortens the operating time, is that this field has technical problem to be solved.
Summary of the invention
The purpose of this section is to summarize some aspects of the embodiment of the present invention and briefly introduce some preferable implementations
Example.It may do a little simplified or be omitted to avoid our department is made in this section and the description of the application and the title of the invention
Point, the purpose of abstract of description and denomination of invention it is fuzzy, and this simplification or omit and cannot be used for limiting the scope of the invention.
In view of above-mentioned technological deficiency, the present invention is proposed.
Therefore, as one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides one kind and mentions
The method of 10 kinds of gibberellin detection stability of high detection simultaneously.
In order to solve the above technical problems, the present invention provides the following technical scheme that it is a kind of improve simultaneously detect 10 kinds it is red mould
The method of element detection stability comprising,
The extraction of 10 kinds of gibberellin: the grinding of crops blade is added and extracts reagent, ultrasound, extraction of ocean eddies；
The purifying of sample: it is cleaned and is enriched with using MAX and C18 (CNW LC-C18200mg)；
It tests and analyzes: being measured using ultra performance liquid chromatography-tandem mass spectrometer, calculate the content of gibberellin to be measured.
A kind of preferred embodiment of method as 10 kinds of gibberellin detection stability of raising of the present invention while detection:
The extraction of 10 kinds of gibberellin, including, by fresh crops blade 200mg through liquid nitrogen grinding, then it is added and contains 0.1% formic acid
70% second eyeball extraction reagent 1.5mL, carry out room temperature ultrasound 5min, vortex 5min extract, used after extraction
The centrifuge separation of 14000rpm revolving speed takes supernatant 1.0mL to volatilize solvent using freeze-drying centrifuge, reuses 70% second eyeball
100uL dissolves sample, adds pure water 700uL and is diluted to the purifying that organic solvent acetonitrile content 10% carries out sample.
A kind of preferred embodiment of method as 10 kinds of gibberellin detection stability of raising of the present invention while detection:
The purifying of the sample, including use MAX and C18 (CNW LC-C18200mg) according to mass ratio 1:(2~4) carry out removal of impurities and
Enrichment.
A kind of preferred embodiment of method as 10 kinds of gibberellin detection stability of raising of the present invention while detection:
The detection and analysis, including using 70% second eyeball to be surveyed as eluant, eluent using ultra performance liquid chromatography-tandem mass spectrometer
It is fixed, calculate the content of gibberellin to be measured.
A kind of preferred embodiment of method as 10 kinds of gibberellin detection stability of raising of the present invention while detection:
It further include replacing solvent as internal standard 2- chlorophenylalanine, in the detection process, passing through 2- chlorophenylalanine peak area and guarantor
Stay the acquisition situation of time reflection actual sample；The 2- chlorophenylalanine concentration is 1 μ g/mL.
A kind of preferred embodiment of method as 10 kinds of gibberellin detection stability of raising of the present invention while detection:
It further include using 4,4 '-di-2-ethylhexylphosphine oxides (2- chloroaniline), P-anisidine, l-tyrosine methyl esters, 3- chloroaniline, 2,4- dimethyl
Quinoline, sulfapryidine, Atrazine, sulfadoxine, DL-leucine, N- benzoyl-l-tyrosine ethyl ester, 6- phenyl -2- sulphur
Urea pyrimidine, N- (toluoyl) glycine, Metronidazole, enoxolone, flavanones, 6-caprolactone,
The mixed sample of 2-aminopyridine is as general quality-control product, and detection is overlapped at least into the general quality-control product of two needles according to spectrogram every time
The state of situation analysis detecting instrument.
A kind of preferred embodiment of method as 10 kinds of gibberellin detection stability of raising of the present invention while detection:
By quality ratio, described 4,4 '-di-2-ethylhexylphosphine oxides (2- chloroaniline): P-anisidine: l-tyrosine methyl esters: 3- chloroaniline: 2,4- bis-
Methylquinoline: sulfapryidine: Atrazine: sulfadoxine: DL-leucine: N- benzoyl-l-tyrosine ethyl ester: 6- phenyl-
2- deracil: N- (toluoyl) glycine: Metronidazole: enoxolone: flavanones: ε-is in oneself
Ester: 2-aminopyridine=1:0.5:0.5:2:1:0.4:0.1:0.1:0.2:0.5:0.5:1:1:0.1:0.5:2:0.2.
A kind of preferred embodiment of method as 10 kinds of gibberellin detection stability of raising of the present invention while detection:
The concentration of the 4,4 '-di-2-ethylhexylphosphine oxide (2- chloroaniline) is 1 μ g/mL, the concentration of P-anisidine is 0.5 μ g/mL, l-tyrosine first
The concentration of ester is 0.5 μ g/mL, the concentration of 3- chloroaniline is 2 μ g/mL, the concentration of 2,4- dimethyl quinoline is 1 μ g/mL, sulfanilamide (SN) pyrrole
The concentration of pyridine is 0.4 μ g/mL, the concentration of Atrazine is 0.1 μ g/mL, the concentration of sulfadoxine is the bright ammonia of 0.1 μ g/mL, DL-
Acid concentration be 0.2 μ g/mL, N- benzoyl-l-tyrosine ethyl ester concentration be 0.5 μ g/mL, 6- phenyl -2- deracil
Concentration be 0.5 μ g/mL, the concentration of N- (toluoyl) glycine is 1 μ g/mL, Metronidazole
Concentration is 1 μ g/mL, the concentration of enoxolone is 0.1 μ g/mL, the concentration of flavanones is 0.5 μ g/mL, the concentration of 6-caprolactone is
2 μ g/mL, 2-aminopyridine concentration be 0.2 μ g/mL.
Beneficial effects of the present invention: the present invention need not individually prepare quality-control product during the experiment, especially when high-volume
When detection, the general quality-control product of the present invention is particularly convenient, and the unstable original of instrument can be gone out according to spectrogram overlapping cases successful analysis
Cause, accuracy of judgement degree reach 100%, meanwhile, the present invention selects 2- chlorophenylalanine as general internal standard, and the accurate sample that monitors is adopted
Collection state, general internal standard that the present invention selects is stable, accurate detection is realized under negative ions mode, to the detection of gibberellin not
Interference can be generated, the pre-treating method impurity in reproducible gibberellin detection method of the present invention of detection significantly reduces, and energy energy
Substantially reduce the pollution to instrument, the present invention detects gibberellin, and to go out peak response more excellent, obviously more than prior art detection sensitivity
It is high.The method of the present invention uses 70% second eyeball of 0.1% formic acid to be mixed the extraction reagent as gibberellin, can improve extraction effect
Rate, GA1, GA3, GA4, GA7, GA9, GA15, GA19, GA20, GA24, GA53 recovery rate respectively reach 93%, 95%, 89%,
83%, 89%, 103%, 98%, 102%, 91%, 95%, RSD is respectively less than 10%；The present invention using ultrasound and be vortexed simultaneously into
Row extracts, and greatly shortens extraction time, amounts to extraction time 10min, extraction efficiency is high and extraction stability is good.
The present invention uses MAX and C18 (CNW LC-C18200mg) SPE pillar as purified material, can be more efficient remove
Miscellaneous and enrichment gibberellin；The present invention extracts and the crop sample of purifying is able to detect that the gibberellin of low concentration, and reduces instrument
The pollution of device enhances the stability of detection.
In the prior art, at the same detect a variety of gibberellin due to by extracting method, purification step, sample acquisition situation,
Instrument fluctuation etc. be affected, therefore often result in detection stability it is bad, the research of the invention finds that, by adjusting in pre-treatment
Extracted using the 70% second eyeball containing 0.1% formic acid as the method extracted reagent, select room temperature ultrasound 5min+ vortex 5min,
Select MAX and C18 (CNW LC-C18200mg) according to mass ratio 1:(2~4) as purified material, and mixed and be denoted as using 17
Sample is monitored as internal standard for general quality-control product monitoring instrument state, 2- chlorophenylalanine and acquires situation, substantially increases this hair
The detection stability of bright gibberellin, detection repeat, and error is small.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, required use in being described below to embodiment
Attached drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for this
For the those of ordinary skill of field, without any creative labor, it can also be obtained according to these attached drawings other
Attached drawing.Wherein:
Fig. 1 is the setting demonstration of the method for the present invention MultiQuant software parameter.
Fig. 2 is the datagram for the sample internal standard exception that the present invention detects.
Fig. 3 is the peak figure out for the exceptional sample that the present invention detects.
Fig. 4 is the peak figure out that the present invention resurveys rear normal sample.
Fig. 5 is quality-control product of the present invention appearance situation map on liquid chromatograph-mass spectrometer device.
Fig. 6 is overlap of peaks situation map of the quality-control product of the present invention on liquid chromatograph-mass spectrometer device.
Fig. 7 is the appearance and overlap of peaks situation map of the mixed mark quality-control product of the present invention in one-time authentication experiment.
Fig. 8 is in one-time authentication experiment, it is known that the appearance and overlap of peaks situation map of sample.
Fig. 9 is the appearance situation map of the mixed mark quality-control product of the present invention in one-time authentication experiment.
Figure 10 is in one-time authentication experiment, it is known that the appearance and overlap of peaks situation map of sample.
Figure 11 is the appearance and overlap of peaks situation map of the mixed mark quality-control product of the present invention in one-time authentication experiment.
Figure 12 is in one-time authentication experiment, it is known that the appearance and overlap of peaks situation map of sample.
Figure 13 is the chromatographic peak comparison that the method for the present invention and prior art processing method detect wheat leaf blade GA content.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, right combined with specific embodiments below
A specific embodiment of the invention is described in detail.
In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention, but the present invention can be with
Implemented using other than the one described here other way, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by the specific embodiments disclosed below.
Secondly, " one embodiment " or " embodiment " referred to herein, which refers to, may be included at least one realization side of the invention
A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to
The same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiments.
Embodiment 1:
The extraction of 10 kinds of gibberellin in sample: taking fresh crops blade 200mg through liquid nitrogen grinding, is then added and contains
The extraction reagent 1.5mL of 70% second eyeball of 0.1% formic acid, carries out room temperature ultrasound 5min, and vortex 5min is extracted, made after extraction
It is centrifugated with 14000rpm revolving speed, takes supernatant 1.0mL to volatilize solvent using freeze-drying centrifuge, reuse 70% second eyeball
100uL dissolves sample, adds pure water 700uL and is diluted to the progress subsequent purification of organic solvent acetonitrile content 10%；
The enrichment and purifying of sample: using MAX and C18 (CNW LC-C18200mg) according to mass ratio 1:(2~4) it carries out
Removal of impurities and enrichment；
Ultra performance liquid chromatography-tandem mass spectrum tests and analyzes: 70% second eyeball being used to use ultra high efficiency liquid as eluant, eluent
Phase chromatography-tandem mass spectrometer is measured, and calculates the content of gibberellin to be measured.
Liquid chromatogram and mass spectrographic experimental setup are as follows:
Liquid phase chromatogram condition:
Chromatographic column: ACQUITY HSS T3 column, i.d.2.1 × 100mm, 1.8um
Mobile phase A: 0.04% acetic acid/ultrapure water；Mobile phase B: 0.04% acetic acid/second eyeball；Wash needle liquid: 50% methanol/water
(ultrasonic degassing 10min after mixing)
Column temperature: 45 DEG C of flow velocitys: 0.35ml/min sample volume: 5uL
Eluent gradient condition
Mass Spectrometry Conditions:
Acquire ion pair information:
Embodiment 2:
Influence of the reagent for extraction efficiency and extraction stability is extracted in 1 method of embodiment and is shown in Table 1, selects 70% respectively
Second eyeball, the 70% second eyeball containing 0.1% formic acid, 80% methanol, 80% methanol containing 0.1% formic acid are as extraction reagent.
Table 1
As known from Table 1, the 70% second eyeball containing 0.1% formic acid extracts simultaneously as the extraction optimal stability for extracting reagent
Efficiency highest.
Influence of the extracting method, temperature of gibberellin for extraction efficiency and extraction stability is shown in Table in 1 method of embodiment
2, room temperature ultrasound 5min+ vortex 5min, room temperature ultrasound 5min, 4 DEG C of low temperature ultrasonic 5min, vortex 5min is respectively adopted and is mentioned
It takes.
Table 2
As known from Table 2, the optimal stability extracted using the method for room temperature ultrasound 5min+ vortex 5min, is mentioned simultaneously
Take efficiency highest.
Influence of the organic solvent acetonitrile content for the gibberellin rate of recovery is shown in Table 3 in embodiment 1, and 10% second is respectively adopted
Nitrile, 20% acetonitrile carry out following purification steps.
Table 3
| GA1 | GA3 | GA4 | GA7 | GA9 | GA15 | GA19 | GA20 | GA24 | GA53 | |
| 10% second eyeball | 88% | 98% | 113% | 92% | 115% | 105% | 84% | 100% | 93% | 94% |
| 20% second eyeball | 80% | 55% | 83% | 82% | 108% | 104% | 80% | 93% | 90% | 92% |
From table 3 it can be seen that the rate of recovery is higher than containing 20% second when organic solvent acetonitrile content 10% when crossing chromatography
Eyeball.
Influence of the model of the chromatographic column used to the repeatability that gibberellin of the present invention extracts is purified in embodiment 1 is shown in Table 4:
Table 4
From table 4, it can be seen that selecting chromatographic column of the present invention, the stability of extraction is more preferably.
To sum up, since GA content is low, situation, instrument are acquired by extracting method, purification step, sample when detecting
Fluctuation etc. be affected, and use the method for the present invention, the method for the present invention use 70% second eyeball of 0.1% formic acid carry out mixing as
The extraction reagent of gibberellin, can improve extraction efficiency, GA1, GA3, GA4, GA7, GA9, GA15, GA19, GA20, GA24, GA53
Recovery rate respectively reaches 93%, 95%, 89%, 83%, 89%, 103%, 98%, 102%, 91%, and 95%, RSD is respectively less than
10%；The present invention is using ultrasound and is vortexed while extracting, and greatly shortens extraction time, amounts to extraction time 10min, extracts
High-efficient and extraction stability is good.
The present invention uses MAX and C18 (CNW LC-C18200mg) SPE pillar as purified material, can be more efficient remove
Miscellaneous and enrichment gibberellin；The present invention extracts and the crop sample of purifying is able to detect that the gibberellin of low concentration, and reduces instrument
The pollution of device enhances the stability of detection.
The pre-treating method and detection method of 10 kinds of gibberellin detection in crops blade of the present invention, 0.02ng/mL~
Linear good in 800ng/mL concentration range, correlation coefficient r can reach 0.99 or more.
0.02~0.05ng/ml of detection limit of the present invention:
Simultaneously as GA content is low, therefore situation, instrument are acquired by extracting method, purification step, sample when detection
Fluctuation etc. is affected, and for the monitoring of sample acquisition situation, the present invention selects 2- chlorophenylalanine as internal standard, is configured to 1 μ
The internal standard extracting solution of g/mL replaces pure solvent to extract experiment sample with internal standard 2- chlorophenylalanine extracting solution, guarantees every
Interior target concentration is consistent in a sample, and upper machine acquisition data are set in data acquisition by MultiQuant software parameter
It sets, checks internal standard peak area and retention time, situation is acquired by interior target, reflects that the acquisition situation of actual sample, internal standard are different
Normal individual samples, then open corresponding acquisition data and check, data exception adopt again.
The setting of MultiQuant software parameter: the other materials in addition to internal standard are deleted；Generate target knot in all samples
Fruit table；It chooses area one to arrange, clicks creates ametric plot, generate area line chart, line chart ordinate is changed
At the form of percentage, addition one represents internal standard area average value horizontal line, sample internal standard area 10% model above and below average value
In enclosing, it is believed that sample data acquisition is normal, returns again to data acquisition software beyond 10% and checks sample collection data, exceptional sample
Resubmit acquisition；It chooses retention time one to arrange, clicks creates a metric plot, generate retention time line chart, partially
Difference is more than that 0.1 returned data acquisition software checks sample collection data, and exceptional sample resubmits acquisition.
The present invention passes through repeated screening from numerous substances, selects 2- chlorophenylalanine as internal standard, finds it positive and negative
Under ion mode can appearance and peak type it is good, and be not easy to test substance generate interference, can using the method for the present invention
The acquisition situation of accurate measurements sample, and will not omit, for example, when detection sample mixes bubble during sample-adding,
It will cause sample volume reduction, cause out peak response reduction, so that the quantitative inaccuracy of the final sample, art methods
This technical problem can not be monitored, after internal standard quality-control product of the present invention, when a certain sample mixes bubble, is added in the sample
The interior target appearance response entered can also reduce, therefore can accurately find the out of condition sample of the acquisition.
And works as and select other substances as internal standard, such as 3- iodotyrosine, 15 fluorine octanoic acids, Sucralose, 3- chlorobenzene
Whens amine, tridecanoic acid, glycine, benzoic acid, succinic acid etc., due to influencing difference too under its is unstable or negative ions mode
Greatly or sample material to be also easy to produce interference, detection repeatability bad, therefore be not suitable as general internal standard.
Fig. 1 is the setting of the method for the present invention MultiQuant software parameter.Fig. 2 is the sample of internal standard exception of the present invention, and Fig. 3 is
The sample goes out peak figure, from Fig. 2 and Fig. 3 it is found that when internal standard is abnormal, illustrates sample collection exception, Fig. 4 is by the sample
Go out peak figure after resurveying sample introduction, comparison diagram 3 illustrates choosing of the present invention it is found that peak response is decreased obviously out when Fig. 3 sample collection
2- chlorophenylalanine, which is selected, as internal standard can accurately reflect sample collection situation.
In order to the fluctuation status of instrument when monitor and detection simultaneously, the present invention screens 17 mixed marks and forms general Quality Control
Product, 17 standard specimens of general quality-control product of the invention and its are shown in Table 1 using final concentration:
Table 1
The present invention selects the compound that separating degree is good, response is suitable, stable and is used as mixed mark, 17 marks by testing repeatedly
Sample appearance time is substantially uniformly distributed in 0~11 minute, mutually smaller on the influence of appearance time each other, is responded in 1*10^6~3*
Between 10^7, belonging to the best response range of used test instrument, 17 standard specimen compound structures are stablized, and it is not volatile, rotten, two
Each substance responds fluctuation is prepared within 20% using hundreds of potential possibility as the substance of standard items respectively between secondary acquisition
At the mother liquor of 1mg/mL and the working solution of 1 μ g/mL, is developed with the liquid matter method that working solution carries out standard items, determines testing conditions,
Again with mother liquor at a hybrid standard product solution, upper machine acquisition, and the concentration of standard items is adjusted, is made into mixed mark, upper machine is adopted
Collect data, causes quality-control product unstable or the detection peak of each standard specimen due to that may react to each other between the standard specimen of various combination
Between be easy to interfere with each other or respond bad etc., therefore the present invention is by combining repeatedly, screening, it is final determine 17 can
Suitable for the mixed mark quality-control product of various different classes of substance detections, 17 mixed mark quality-control product chromatographies go out peak figure as shown in figure 5, from figure
17 standard specimen detection peaks are separated from each other known to 5, are not interacted substantially, and respond well, in the most suitable range of instrument, 17
A mixed mark total ion chromatogram overlay chart is as shown in Figure 6.The mixed mark quality-control product overlapping of two needles is fine as can be seen from Figure 6, peak height
Unanimously, show that instrument response is stablized, retention time is consistent, shows that chromatographic isolation situation is stablized, instrument state is good.Matter of the present invention
Control product can be used as general quality-control product, the detection quality-control product suitable for all substances.
Judge that instrument fluctuates by the data total ion chromatogram overlapping cases that front and back acquires in data acquisition
Situation, chromatogram overlapping preferably illustrate instrument stabilizer, and overlapping is bad then to judge instrument state by overlapping cases, such as when reservation
Between be not overlapped explanation may be column pressure or column temperature it is unstable, peak height be overlapped not Shang explanation may be chromatographic column block or mass spectrum dirt
Dye etc..
The mixed mark quality-control product of the present invention is sufficiently stable, and prepares simply, and dosage is few, at low cost.The present invention passes through for difference
The multiplicating of kind of substance, which detects, to be proved, being capable of accurately reaction chromatography instrument or mass spectrometric instrument using quality-control product of the present invention
State (whether instrument stable, column pressure, whether column temperature is stable, whether pillar has blocking, whether has pollution etc.) and sample to be tested
State, and the unstable reason of instrument can be gone out according to spectrogram overlapping cases successful analysis, accuracy of judgement degree reaches 100%.
Using the known sample of different conditions as standard, to verify quality-control product of the invention for different sample states and instrument
The monitoring accuracy of device state, each known sample do primary repetition sample introduction, each mixed mark quality-control product, which is done, once repeats sample introduction,
If the appearance and overlapping cases of known sample are consistent with the appearance of the mixed mark quality-control product of the present invention and overlapping cases, illustrate the present invention
The case where mixed mark quality-control product is for sample and instrument state reflection is accurate.It is verified by many experiments, finds the mixed mark matter of the present invention
Control product can 100% reflected sample acquisition situation and when sampling instrument operating status, by the appearance of mixed mark quality-control product and again
Folded situation, can reflect the acquisition situation of sample to be tested.
As shown in Figure 7 and Figure 8, Fig. 7 is the appearance and overlap of peaks feelings of the mixed mark quality-control product of the present invention in one-time authentication experiment
Condition, as shown in fig. 7, the mixed mark quality-control product overlapping of two needles is very well, peak height is consistent, shows that instrument response is stablized, retention time is consistent, table
Bright chromatographic isolation situation is stablized, and instrument state is good, as shown in figure 8, in this confirmatory experiment, it is known that the appearance of sample and again
It is folded same fine, illustrate that the mixed mark quality-control product of the present invention is capable of the normal condition of accurate reaction kit.
As shown in Figure 9 and Figure 10, Fig. 9 is the appearance situation of the mixed mark quality-control product of the present invention, from figure in one-time authentication experiment
9 find out, quality-control product peak type is bad, separation is bad, and the mixed mark non-appearance of quality-control product of 10min or so, illustrate chromatographic apparatus when detection
There are problems.Figure 10 is in this confirmatory experiment, it is known that the appearance and overlapping cases of sample, as can be seen from Figure 10, it is known that sample is same
Sample separation is bad, and 8~10min is responded and is lower, and quality-control product of the present invention and known sample response are consistent, and by verifying,
There is problem when detecting in chromatograph.Illustrate that the mixed mark quality-control product of the present invention is capable of the abnormality of accurate reaction kit.
As is illustrated by figs. 11 and 12, Figure 11 is the appearance and peak weight of the mixed mark quality-control product of the present invention in one-time authentication experiment
Folded situation, as shown in figure 11, the mixed mark quality-control product overlap of peaks of two needles are bad, and the offset of 1~3min retention time illustrates to supervise time keeping instrument
Unstable, Figure 12 is in this confirmatory experiment, it is known that the appearance and overlapping cases of sample, as can be seen from Figure 12, it is known that sample is same
Overlap of peaks is bad, and 1~3min sample retention time equally exists offset, and by verifying, and column pressure is not when detecting for chromatograph
Surely, illustrate that the mixed mark quality-control product of the present invention is capable of the abnormality of accurate reaction kit.
The present invention need not individually prepare quality-control product during the experiment, and especially when mass detection, the present invention is general
Quality-control product is particularly convenient, and the unstable reason of instrument can be gone out according to spectrogram overlapping cases successful analysis, and accuracy of judgement degree reaches
100%, meanwhile, the present invention selects 2- chlorophenylalanine as general internal standard, accurate to monitor sample collection state, present invention selection
General internal standard is stable, realizes accurate detection under negative ions mode, will not generate interference, detection weight to the detection of gibberellin
Pre-treating method impurity in the good gibberellin detection method of the present invention of renaturation significantly reduces, and can substantially reduce the dirt to instrument
Dye, Figure 13 is the chromatographic peak comparison that the method for the present invention and prior art processing method detect wheat leaf blade GA content, from figure
13 as can be seen that present invention detection gibberellin to go out peak response more excellent, it is more considerably higher than prior art detection sensitivity.
The method of the present invention uses 70% second eyeball of 0.1% formic acid to be mixed the extraction reagent as gibberellin, can improve
Extraction efficiency, GA1, GA3, GA4, GA7, GA9, GA15, GA19, GA20, GA24, GA53 recovery rate respectively reach 93%, 95%,
89%, 83%, 89%, 103%, 98%, 102%, 91%, 95%, RSD is respectively less than 10%；The present invention is using ultrasound and is vortexed
It extracts simultaneously, greatly shortens extraction time, amount to extraction time 10min, extraction efficiency is high and extraction stability is good.
The present invention uses MAX and C18 (CNW LC-C18200mg) SPE pillar as purified material, can be more efficient remove
Miscellaneous and enrichment gibberellin；The present invention extracts and the crop sample of purifying is able to detect that the gibberellin of low concentration, and reduces instrument
The pollution of device enhances the stability of detection.
In the prior art, at the same detect a variety of gibberellin due to by extracting method, purification step, sample acquisition situation,
Instrument fluctuation etc. be affected, therefore often result in detection stability it is bad, the research of the invention finds that, by adjusting in pre-treatment
Extracted using the 70% second eyeball containing 0.1% formic acid as the method extracted reagent, select room temperature ultrasound 5min+ vortex 5min,
Select MAX and C18 (CNW LC-C18 200mg) according to mass ratio 1:(2~4) as purified material, and mixed and be denoted as using 17
Sample is monitored as internal standard for general quality-control product monitoring instrument state, 2- chlorophenylalanine and acquires situation, substantially increases this hair
The detection stability of bright gibberellin, detection repeat, and error is small.
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to preferable
Embodiment describes the invention in detail, those skilled in the art should understand that, it can be to technology of the invention
Scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered in this hair
In bright scope of the claims.
Claims (4)
1. a kind of method of 10 kinds of gibberellin detection stability of raising while detection, it is characterised in that: including,
The extraction of 10 kinds of gibberellin: by fresh 200 mg of crops blade through liquid nitrogen grinding, 70% second for containing 0.1 % formic acid is added
1.5 mL of extraction reagent of nitrile carries out 5 min of room temperature ultrasound, and vortex 5min is extracted, and is centrifugated using 14000 rpm revolving speeds,
It takes 1.0 mL of supernatant to volatilize solvent in freeze-drying centrifuge, reuses 70% acetonitrile, 100 μ L dissolution sample, it is dilute that pure water is added
It releases to organic solvent acetonitrile content 10% and carries out the purifying of sample；
The purifying of sample: using MAX and C18 column according to mass ratio 1:(2 ~ 4) it is cleaned and is enriched with；The C18 column, specification
For CNW LC-C18 200mg；
It tests and analyzes: being measured using ultra performance liquid chromatography-tandem mass spectrometer, calculate the content of gibberellin to be measured；
Mass spectrographic ion pair information setting are as follows: GA1-2:Q1:347, Q3:259, retention time 3.92, DP-60, CE-22；GA1-
4:Q1:347, Q3:241, retention time 3.92, DP-60, CE-25；GA3-1:Q1:345, Q3:143, retention time 3.88, DP-
60,CE-32；GA3-3:Q1:345, Q3:239, retention time 3.88, DP-60, CE-19；GA4-1:Q1:331, Q3:212.8,
Retention time 6.27, DP-60, CE-41；GA4-2:Q1:331, Q3:257, retention time 6.27, DP-60, CE-30；GA7-2:
Q1:329.2, Q3:211, retention time 6.17, DP-70, CE-34；GA7-3:Q1:329.2, Q3:223, retention time 6.17,
DP-60,CE-24；GA9-1:Q1:315.1, Q3:270.9, retention time 7.38, DP-80, CE-30；GA9-2:Q1:315.1,
Q3:253.3, retention time 7.38, DP-80, CE-32；GA15-1:Q1:329.1, Q3:257.1, retention time 7.36, DP-
75,CE-34；GA15-4:Q1:329.1, Q3:131, retention time 7.36, DP-75, CE-40；GA19-2:Q1:361.2, Q3:
273.2, retention time 4.9, DP-80, CE-37；GA19-6:Q1:361.2, Q3:317.1, retention time 4.9, DP-80, CE-
32；GA20-1:Q1:331.3, Q3:287, retention time 5.11, DP-60, CE-29；GA20-3:Q1:331.3, Q3:243, guarantor
Stay time 5.11, DP-58, CE-28；GA24-1:Q1:345.1, Q3:257.1, retention time 6.54, DP-73, CE-35；
GA24-5:Q1:345.1, Q3:301.1, retention time 6.54, DP-60, CE-30；GA53-2:Q1:347.1, Q3:188.9, guarantor
Stay time 5.73, DP-70, CE-45；GA53-5:Q1:347.1, Q3:303.1, retention time 5.73, DP-70, CE-36；
It further include using 4,4 '-di-2-ethylhexylphosphine oxides (2- chloroaniline), P-anisidine, l-tyrosine methyl esters, 3- chloroaniline, 2,4- bis-
Methylquinoline, sulfapryidine, Atrazine, sulfadoxine, DL-leucine, N- benzoyl-l-tyrosine ethyl ester, 6- phenyl-
2- deracil, N- (toluoyl) glycine, Metronidazole, enoxolone, flavanones, ε-are in oneself
Ester, 2-aminopyridine mixed sample as general quality-control product, detection is at least into the general quality-control product of two needles every time, according to spectrogram weight
The state of folded situation analysis detecting instrument；The 4,4 '-di-2-ethylhexylphosphine oxide (2- chloroaniline): P-anisidine: l-tyrosine methyl esters: 3-
Chloroaniline: 2,4- dimethyl quinolines: sulfapryidine: Atrazine: sulfadoxine: DL-leucine: N- benzoyl-L- junket ammonia
Acetoacetic ester: 6- phenyl -2- deracil: N- (toluoyl) glycine: Metronidazole: Radix Glycyrrhizae time
Acid: flavanones: 6-caprolactone: 2-aminopyridine=1:0.5:0.5:2:1:0.4:0.1:0.1:0.2:0.5:0.5:1:1:0.1:
0.5:2:0.2.
2. the method as described in claim 1 improved while 10 kinds of gibberellin of detection detect stability, it is characterised in that: described
It tests and analyzes, including using 70% acetonitrile to be measured as eluant, eluent using ultra performance liquid chromatography-tandem mass spectrometer, meter
Calculate the content of gibberellin to be measured.
3. the method as claimed in claim 1 or 2 improved while 10 kinds of gibberellin of detection detect stability, it is characterised in that:
It further include replacing solvent as internal standard 2- chlorophenylalanine, in the detection process, passing through 2- chlorophenylalanine peak area and guarantor
Stay the acquisition situation of time reflection actual sample；The 2- chlorophenylalanine concentration is 1 μ g/mL.
4. the method as described in claim 1 improved while 10 kinds of gibberellin of detection detect stability, it is characterised in that: described
The concentration of 4,4 '-di-2-ethylhexylphosphine oxides (2- chloroaniline) is 1 μ g/mL, the concentration of P-anisidine is 0.5 μ g/mL, l-tyrosine methyl esters
Concentration is 0.5 μ g/mL, the concentration of 3- chloroaniline is 2 μ g/mL, the concentration of 2,4- dimethyl quinoline is 1 μ g/mL, sulfapryidine
Concentration is 0.4 μ g/mL, the concentration of Atrazine is 0.1 μ g/mL, the concentration of sulfadoxine is 0.1 μ g/mL, DL-leucine
Concentration be 0.2 μ g/mL, N- benzoyl-l-tyrosine ethyl ester concentration be 0.5 μ g/mL, 6- phenyl -2- deracil it is dense
Degree be 0.5 μ g/mL, the concentration that the concentration of N- (toluoyl) glycine is 1 μ g/mL, Metronidazole
Concentration for 1 μ g/mL, enoxolone is 0.1 μ g/mL, the concentration of flavanones is 0.5 μ g/mL, the concentration of 6-caprolactone is 2 μ g/
ML, 2-aminopyridine concentration be 0.2 μ g/mL.
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| CN114894937A (en) * | 2022-05-30 | 2022-08-12 | 山东诺正检测有限公司 | Method for detecting gibberellic acid in soil |
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