CN113049705A - UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum - Google Patents

UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum Download PDF

Info

Publication number
CN113049705A
CN113049705A CN202110295681.9A CN202110295681A CN113049705A CN 113049705 A CN113049705 A CN 113049705A CN 202110295681 A CN202110295681 A CN 202110295681A CN 113049705 A CN113049705 A CN 113049705A
Authority
CN
China
Prior art keywords
tre
uplc
fusarium graminearum
column
standard
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202110295681.9A
Other languages
Chinese (zh)
Other versions
CN113049705B (en
Inventor
吴琴燕
庄义庆
梁红芳
吴雨琦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhenjiang Institute of Agricultural Sciences Jiangsu Hilly Area
Original Assignee
Zhenjiang Institute of Agricultural Sciences Jiangsu Hilly Area
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhenjiang Institute of Agricultural Sciences Jiangsu Hilly Area filed Critical Zhenjiang Institute of Agricultural Sciences Jiangsu Hilly Area
Priority to CN202110295681.9A priority Critical patent/CN113049705B/en
Publication of CN113049705A publication Critical patent/CN113049705A/en
Application granted granted Critical
Publication of CN113049705B publication Critical patent/CN113049705B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

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
    • 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
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • 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
    • 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
    • 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
    • G01N2030/042—Standards
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a UPLC-MS/MS detection method of T6P and Tre in fusarium graminearum.A sample to be detected is pretreated to prepare a sample solution containing T6P and Tre, the sample solution is eluted and separated by a chromatographic column, data are collected by using an MRM mode of UPLC-MS/MS, an electrospray ion source and a negative ion scanning mode are used for monitoring, the obtained spectrogram of the sample to be detected is compared with a standard spectrogram, corresponding substances are obtained when the retention time is the same, and the T6P and the Tre are respectively quantified by a standard curve linear regression equation; an amino column and a T3 chromatographic column are selected to be suitable for UPLC-MS/MS detection of two compounds of T6P and Tre, a method for simultaneously detecting T6P and Tre in fusarium graminearum by utilizing the two chromatographic columns is respectively established, and the amino column is found to have high sensitivity, the T3 chromatographic column has high recovery rate and good stability. And two chromatographic columns are used for detecting that the content of T6P in the fusarium graminearum pH-1 is respectively about 52.6 mu g/g and 60.5 mu g/g, and the content of Tre is about 4.42mg/g and 5.4 mg/g.

Description

UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum
Technical Field
The invention relates to the field of UPLC-MS/MS detection methods, in particular to a UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum.
Background
Fusarium graminearum (f.graminearum) is the most common pathogenic bacterium in crops such as wheat and corn, and the pathogenic bacterium causes not only the decrease of crop yield, but also the production of vomitoxin, and seriously affects the quality and safety of agricultural products. The biosynthesis pathway of trehalose plays a crucial role in pathogenicity of fungi, and it is reported that related genes of the trehalose synthesis pathway are knocked out from fusarium graminearum, so that the fusarium graminearum loses the trehalose synthesis ability, and then cannot produce spores, and the synthesis ability of vomitoxin is reduced by 86%.
The T6P, 6-trehalose phosphate is an intermediate for Tre and trehalose biosynthesis, is an essential signal metabolite in plants and fungi, quantifies T6P and Tre in Fusarium graminearum, and can provide support for the research of trehalose synthesis path for Fusarium graminearum growth and toxin production control.
Tre belongs to carbohydrate, and at present, a mature detection method exists. T6P belongs to acidic strong sugar, has extremely low content in microorganisms, is mainly detected by GC-MS and LC-MS at present, but has the following defects: GC-MS requires a derivatization reaction, the pretreatment is complex, the detection time is long, the LC-MS detection time is short, the sample pretreatment is simple, but single-rod mass spectrometry scanning is mainly adopted, the parent ions are qualitative and quantitative, and the false positive probability is high. In addition, due to the strong acid property of T6P, the requirement for chromatographic columns is high for compounds with strong polarity which are difficult to be preserved and separated, Dionex IonPac AS11-HC column (250 × 2.0mm, particle size9 μm), SIELC Primese SB column (4.6 × 150mm, 5 μm), water acquisition BEH amide column (3.0 × 100mm,1.7 μm) and ZIC-HILIC HPLC column (2.1 × 150mm,3.5 μm) are reported to be all used for detecting sugar phosphate, except that water acquisition BEH amide column can achieve larger separation degree and has better separation effect, and other chromatographic columns have the problems of low separation degree, low chromatographic response value, short service life of chromatographic columns, and the like.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the problems in the prior art, the invention provides the UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum, which has the advantages of good separation degree, high sensitivity, high recovery rate and good stability.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the following technical scheme:
a UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum specifically comprises the following steps: firstly, preprocessing a to-be-detected sample fusarium graminearum to prepare fusarium graminearum, performing gradient elution separation by a chromatographic column, acquiring data by using an MRM mode of UPLC-MS/MS, monitoring by using an electrospray ion source in a negative ion scanning mode, comparing an obtained spectrogram of the to-be-detected sample with a standard spectrogram, determining corresponding substances with the same retention time, and quantifying T6P and Tre by using a standard curve linear regression equation;
the chromatographic column selects Phenomenexluluna
Figure BDA0002984263080000025
Figure BDA0002984263080000022
(100X 2mm, 3 μm) chromatography Column or a waters Acquity UPLC HSS T3 Column (100X 3mm, 1.7 μm) chromatography Column.
Furthermore, the pretreatment method of the fusarium graminearum sample to be detected comprises the following steps:
placing Fusarium graminearum to be detected in a mung bean soup liquid culture medium, culturing for 7 days at 28 ℃ and 140rpm, filtering to remove mycelia, centrifuging and concentrating to obtain 1 × 105Spore liquid per mL for later use; inoculating spore liquid into potato liquid culture medium, inoculating 1mL of spore liquid into each bottle, shake culturing at 28 deg.C and 140rpm for 7d, filtering to obtain mycelium,washing with deionized water for 3 times, drying, and storing at-20 deg.C;
accurately weighing 0.04g of hypha sample in a 15mL centrifuge tube, adding 4mL deionized water, soaking for 1h, homogenizing by a high-speed homogenizer, adding 4mL methanol, freezing at-80 ℃, ultrasonically thawing and breaking the wall for 30min in an ultrasonic cleaner, centrifuging, and taking the supernatant to be tested.
Furthermore, the mass spectrum conditions are specifically as follows: an ion source: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the detection mode is as follows: MRM multiple reaction monitoring Gas Curtain pressure (Curtain Gas:40 Kpa); electrospray Voltage (iosspray Voltage): -4500 Kpa; ion source Temperature (Temperature): at 450 ℃; atomization Gas pressure (Ion Source Gas 1): 65 Kpa; heat assist Gas pressure (Ion Source Gas 2): 65 Kpa; residence time: 40 ms.
Further, when the detection object is T6P, DP is-110 and CE is-35, -35.
Further, when the detection object is Tre, DP is-95, CE is-19, -25.
Further, the chromatographic column is Phenomenexlulna
Figure BDA0002984263080000026
Figure BDA0002984263080000024
For a (100X 2mm, 3 μm) column, the UPLC optimization conditions were as follows:
mobile phase A: one ten thousandth of ammonia water;
mobile phase B: acetonitrile;
gradient: isocratic elution with 70% A;
flow rate: 0.4 mL/min.
Further, when the Column is a waters Acquity UPLC HSS T3 Column (100 × 3mm, 1.7 μm), the UPLC optimization conditions are as follows:
mobile phase A: 5mM ammonium acetate (containing one ten thousandth of ammonia);
mobile phase B: acetonitrile;
gradient: 99% A at 0-3min, 10% A at 3-5min, and 99% A at 5-8 min;
flow rate: 0.25 mL/min.
Further, the method for acquiring the T6P standard spectrogram specifically comprises the following steps:
weighing a T6P standard substance in a volumetric flask, dissolving the standard substance by using acetonitrile with volume fraction of 60%, fixing the volume, diluting the solution by using the acetonitrile with volume fraction of 60% to prepare standard working solutions with a plurality of concentration gradients step by step, carrying out UPLC-MS/MS analysis on the standard working solutions with different concentrations, and making a standard curve and a spectrogram.
Further, the method for acquiring the Tre standard spectrogram specifically comprises the following steps:
weighing a Tre standard substance in a volumetric flask, dissolving the Tre standard substance by using acetonitrile with the volume fraction of 60% and fixing the volume, then diluting the Tre standard substance by using the acetonitrile with the volume fraction of 60% step by step to prepare a plurality of standard working solutions with concentration gradients, carrying out UPLC-MS/MS analysis on the standard working solutions with different concentrations, and making a standard curve and a spectrogram.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the selected chromatographic column has the advantages of good separation degree, high sensitivity, high recovery rate and good stability.
(2) The pretreatment method is simple and efficient, and the separation effect is further improved.
Drawings
FIG. 1 is a graph showing a comparison of the response intensities of two different columns shown in the MRM chromatogram of a T6P standard solution;
FIG. 2 is a graph showing a comparison of the response intensities of two different columns as shown in the MRM chromatogram of a Tre standard solution;
FIG. 3 is a graph showing the effect of ammonia on T6P response values in T3 column detection vs. chromatogram;
FIG. 4 is a schematic diagram of a standard curve for amino column separation T6P;
FIG. 5 is a schematic diagram of a standard curve for T6P for T3 column separation;
FIG. 6 is a schematic diagram of a standard curve for an amino column separation Tre;
fig. 7 is a standard curve diagram of T3 chromatographic column for separation of Tre.
Detailed Description
The present invention is further illustrated by the following specific examples.
The instruments and reagent sources used in the examples of the invention are as follows:
agilent model 1290 UPLC system, ABsciex 4500 mass spectrometer detector;
an electronic balance: model XP105DR — sartorius scientific instruments ltd;
AWL-020I-P type ultrapure water system-Aikopu Instrument Co., Ltd;
KQ-250E ultrasonic cleaner-Kunshan Selengensis ultrasonic Instrument Co., Ltd;
H2050R type medical centrifuge, Hunan instrument laboratory Instrument development Co., Ltd;
DW-86L828J model ultra-low temperature refrigerator-Haier biomedical products Co., Ltd;
vortex mixer-Jiangsu Haimen Lin Beier Instrument Co.
Methanol and acetonitrile are chromatographically pure, produced by merck corporation of america; ammonium acetate is more than or equal to 98 percent, produced by Germany CNW company; T6P and Tre were purchased from sigma-aldrich trade company, Mass Spectroscopy using distilled water as Drech.
The pretreatment method of the sample to be detected in the embodiment and the comparative example comprises the following steps:
placing Fusarium graminearum to be detected in a mung bean soup liquid culture medium, culturing for 7 days at 28 ℃ and 140rpm, filtering to remove mycelia, centrifuging and concentrating to obtain 1 × 105Spore liquid per mL for later use; inoculating the spore liquid into a potato liquid culture medium, inoculating 1mL of the spore liquid into each bottle, performing shake culture at 28 ℃ and 140rpm for 7d, filtering to obtain hyphae, cleaning for 3 times by using deionized water, drying, and storing at-20 ℃ to be detected;
accurately weighing 0.04g of hypha sample in a 15mL centrifuge tube, adding 4mL deionized water, soaking for 1h, homogenizing by a high-speed homogenizer, adding 4mL methanol, freezing at-80 ℃, ultrasonically thawing and breaking the wall for 30min in an ultrasonic cleaner, centrifuging, and taking the supernatant to be tested.
The method for acquiring the standard spectrogram and the standard curve in the embodiment and the comparative example is as follows:
the method for acquiring the T6P standard spectrogram specifically comprises the following steps:
weighing a T6P standard substance in a volumetric flask, dissolving the standard substance by using acetonitrile with volume fraction of 60%, fixing the volume, diluting the solution by using the acetonitrile with volume fraction of 60% to prepare standard working solutions with a plurality of concentration gradients step by step, carrying out UPLC-MS/MS analysis on the standard working solutions with different concentrations, and making a standard curve and a spectrogram. The standard curve is shown in fig. 4 and 5.
The method for acquiring the Tre standard spectrogram specifically comprises the following steps:
weighing a Tre standard substance in a volumetric flask, dissolving the Tre standard substance by using acetonitrile with the volume fraction of 60% and fixing the volume, then diluting the Tre standard substance by using the acetonitrile with the volume fraction of 60% step by step to prepare a plurality of standard working solutions with concentration gradients, carrying out UPLC-MS/MS analysis on the standard working solutions with different concentrations, and making a standard curve and a spectrogram. The standard curve is shown in fig. 5 and fig. 6.
It can be seen by plotting the standard curve:
when T6P and Tre pass through the amino column, the linear relation is good in the linear range of 4.25-250ug/L, and when the T3 column passes through the linear relation is good in the linear range of 31.25-1000 ug/L; in addition, the detection limit of the amino column is 1.25-5.10 ug/L, which is far lower than that of T3 column, 15.62-12.75 ug/L; the quantitative limit amino column is 3.75-15.60ug/L and is also lower than 38.25-52.03ug/L of the T3 column, the sensitivity of the amino column is higher than that of the T3 column, the correlation coefficients of the two chromatographic columns are both more than 0.99, and the correlation coefficients meet the requirements of actual analysis.
TABLE 1 Standard Curve, Linear Range, correlation coefficient, detection Limit and quantitation Limit for T6P and Tre
Figure BDA0002984263080000041
Example 1:
the UPLC-MS/MS detection method of T6P in fusarium graminearum specifically comprises the following steps: firstly, pretreating a to-be-detected sample fusarium graminearum to prepare the to-be-detected sample fusarium graminearum, eluting and separating the to-be-detected sample fusarium graminearum through a chromatographic column, acquiring data by using an MRM mode of UPLC-MS/MS, monitoring by using an electrospray ion source in a negative ion scanning mode, comparing an obtained spectrogram of the to-be-detected sample with a standard spectrogram, determining corresponding substances with the same retention time, and quantifying T6P through a standard curve linear regression equation;
the chromatographic column selects Phenomenexluluna
Figure BDA0002984263080000053
Figure BDA0002984263080000052
(100X 2mm, 3 μm) column: the UPLC optimization conditions were as follows:
mobile phase A: one ten thousandth of ammonia water;
mobile phase B: acetonitrile;
gradient: isocratic elution with 70% A;
flow rate: 0.4 mL/min.
The mass spectrum conditions are specifically as follows: an ion source: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the detection mode is as follows: MRM multiple reaction monitoring Gas Curtain pressure (Curtain Gas:40 Kpa); electrospray Voltage (iosspray Voltage): -4500 Kpa; ion source Temperature (Temperature): at 450 ℃; atomization Gas pressure (Ion Source Gas 1): 65 Kpa; heat assist Gas pressure (Ion Source Gas 2): 65 Kpa; residence time: 40 ms. DP was-110 and CE was-35, -35.
Example 2:
the UPLC-MS/MS detection method of T6P in fusarium graminearum specifically comprises the following steps: firstly, preprocessing a sample to be detected, namely, preparing fusarium graminearum, performing gradient elution separation by a chromatographic column, acquiring data by using an MRM mode of UPLC-MS/MS, monitoring by using an electrospray ion source in a negative ion scanning mode, comparing an obtained spectrogram of the sample to be detected with a standard spectrogram, determining corresponding substances with the same retention time, and quantifying T6P by using a standard curve linear regression equation;
the chromatographic Column is a waters Acquity UPLC HSS T3 Column (100X 3mm, 1.7 μm): the UPLC optimization conditions were as follows:
mobile phase A: 5mM ammonium acetate (containing ten-thousandths of ammonia);
mobile phase B: acetonitrile;
gradient: 99% A at 0-3min, 10% A at 3-5min, and 99% A at 5-8 min;
flow rate: 0.25 mL/min.
The mass spectrum conditions are specifically as follows: an ion source: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the detection mode is as follows: MRM multiple reaction monitoring Gas Curtain pressure (Curtain Gas:40 Kpa); electrospray Voltage (iosspray Voltage): -4500 Kpa; ion source Temperature (Temperature): at 450 ℃; atomization Gas pressure (Ion Source Gas 1): 65 Kpa; heat assist Gas pressure (Ion Source Gas 2): 65 Kpa; residence time: 40 ms. DP was-110 and CE was-35, -35.
Example 3:
the UPLC-MS/MS detection method for Tres in fusarium graminearum specifically comprises the following steps: firstly, pretreating a to-be-detected sample fusarium graminearum to prepare the to-be-detected sample fusarium graminearum, eluting and separating the to-be-detected sample fusarium graminearum through a chromatographic column, acquiring data by using an MRM mode of UPLC-MS/MS, monitoring by using an electrospray ion source and a negative ion scanning mode, comparing an obtained spectrogram of the to-be-detected sample with a standard spectrogram, determining corresponding substances with the same retention time, and quantifying Tre through a standard curve linear regression equation;
the chromatographic column selects Phenomenexluluna
Figure BDA0002984263080000063
Figure BDA0002984263080000062
(100X 2mm, 3 μm) column: the UPLC optimization conditions were as follows:
mobile phase A: one ten thousandth of ammonia water;
mobile phase B: acetonitrile;
gradient: isocratic elution with 70% A;
flow rate: 0.4 mL/min.
The mass spectrum conditions are specifically as follows: an ion source: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the detection mode is as follows: MRM multiple reaction monitoring Gas Curtain pressure (Curtain Gas:40 Kpa); electrospray Voltage (iosspray Voltage): -4500 Kpa; ion source Temperature (Temperature): at 450 ℃; atomization Gas pressure (Ion Source Gas 1): 65 Kpa; heat assist Gas pressure (Ion Source Gas 2): 65 Kpa; residence time: 40 ms; DP was-95 and CE was-19, -25.
Example 4:
the UPLC-MS/MS detection method for Tres in fusarium graminearum specifically comprises the following steps: firstly, preprocessing a sample to be detected, namely, preparing fusarium graminearum, performing gradient elution separation by a chromatographic column, acquiring data by using an MRM mode of UPLC-MS/MS, monitoring by using an electrospray ion source and in a negative ion scanning mode, comparing an obtained spectrogram of the sample to be detected with a standard spectrogram, determining corresponding substances when retention time is the same, and quantifying Tre by using a standard curve linear regression equation;
the chromatographic Column is a waters Acquity UPLC HSS T3 Column (100X 3mm, 1.7 μm): the UPLC optimization conditions were as follows:
mobile phase A: 5mM ammonium acetate (containing ten-thousandths of ammonia);
mobile phase B: acetonitrile;
gradient: 99% A at 0-3min, 10% A at 3-5min, and 99% A at 5-8 min;
flow rate: 0.25 mL/min.
The mass spectrum conditions are specifically as follows: an ion source: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the detection mode is as follows: MRM multiple reaction monitoring Gas Curtain pressure (Curtain Gas:40 Kpa); electrospray Voltage (iosspray Voltage): -4500 Kpa; ion source Temperature (Temperature): at 450 ℃; atomization Gas pressure (Ion Source Gas 1): 65 Kpa; heat assist Gas pressure (Ion Source Gas 2): 65 Kpa; residence time: 40 ms; DP was-95 and CE was-19, -25.
Comparative examples 1 to 4:
other embodiments of comparative examples 1-4 are the same as examples 1-2, except that the selection and optimization conditions of the column are different, as shown in table 2:
table 2 selection of columns and UPLC condition optimization for the testing procedure of examples 1-2 and comparative examples 1-4T6P
Figure BDA0002984263080000071
As can be seen from table 2: in comparative examples 1-4, HILIC and Cogent Diamond Hydride were strongly retained without a peak; when a C18 chromatographic column is adopted, the retention of T6P is weak, and T6P flows out randomly at about 2 min; the water Acquity BEH amide column has large pressure and trailing phosphate sugar peak, after a chromatographic column uses 200-needle samples, phosphate sugar is strongly reserved, and the chromatographic column cannot be continuously used for detecting phosphate sugar; in contrast, the luna amino silica gel column and the waters Acquity UPLC HSS T3 chromatographic columns respectively used in embodiments 1 and 2 of the present application have a good retention effect of T6P, the response intensity of T6P of 50 μ g/L detected by the amino column is about 3.5 × 104cps, while the response value of T6P of 1000 μ g/L detected by the T3 chromatographic column is only 1 × 104cps, and the response intensity of T6P detected by the T3 chromatographic column is much lower than that of the amino column, which can be specifically referred to the MRM chromatogram in fig. 1, wherein the concentration of T6P of the amino column is 50 μ g/L, the concentration of T6P of the T3 chromatographic column is 1000 μ g/L, the dashed ion pair is 421.1/241.1, and the solid ion pair is 421.1/79.1.
Comparative examples 5 to 8:
other embodiments of comparative examples 5-8 are the same as examples 3-4, except that the selection and optimization conditions of the columns are different, as shown in table 3:
table 3 selection and UPLC condition optimization of chromatography columns for the Tre detection process of examples 3-4 and comparative examples 1-4
Figure BDA0002984263080000081
As can be seen from table 3: in addition to the C18 column, other columns can achieve separation of Tre. However, KinetexHilic, code Diamond Hydride and waters acquisition BEH amide column show strong retention in T6P separation, and these 3 columns cannot achieve simultaneous detection of T6P and Tre. T3 and NH2 chromatographic columns can realize the simultaneous detection of T6P and Tre.
Reference may be made in particular to the MRM chromatogram of FIG. 2, in which the Tre concentration is 50. mu.g/L, the ion pair in dotted line is 341.0/178.8, and the ion pair in solid line is 341.0/118.9.
In addition, the optimization experiment for the T3 column test T6P for example 5 and examples 1-2 is shown in FIG. 3: color development in example 5When the spectrum column optimizes conditions: ten-thousandth of ammonia water is added into the water phase. The response of T6P was found to be improved ten-fold, but the response value was still much lower than that of the NH3 column. Luna
Figure BDA0002984263080000082
The column efficiency loss is fast, the average life of the detection of the phosphoglucose by one chromatographic column is 200-300 samples, the service life of the T3 column is long, and the response value is low, so that the detection cost and the content of T6P in the sample are comprehensively considered, and a proper chromatographic column is selected for determination. In FIG. 3, the concentration of T6P was 1000. mu.g/L, and the ion pair was 421.1/79.1.
In addition, the recovery and precision of examples 1 to 4 were measured.
The method comprises the steps of accurately weighing 40mg of fusarium graminearum, repeating each concentration for 4 times after adding a standard solution with a certain concentration, and calculating the recovery rate and the Relative Standard Deviation (RSD) of the method, wherein the results show that the recovery rates of T6P are 71.5% -85.7% and 83.7% -95.6% respectively and the relative standard deviations are 3.8% -6.2% and 2.7% -3.3% under 3 standard adding levels by utilizing two chromatographic columns of amino and T3; the recovery rates of Tre are 72.4% -82.3% and 82.8% -84.8%, and the relative standard deviation is 4.5% -8.5% and 2.8% -3.2%; compared with the amino column, the recovery rate of the 3 column is high, and the RSD is low, which indicates that the stability of the T3 column is superior to that of the amino column. The amino and T3 chromatographic columns are used for detecting that the content of T6P in the fusarium graminearum pH-1 is about 52.6 mu g/g and 60.5 mu g/g respectively, the content of Tre is about 4.42mg/g and 5.4mg/g, and the two chromatographic columns are used for detecting that the content of T6P and the content of Tre in the fusarium graminearum pH-1 have no significant difference and can be used for detecting the content of T6P and the content of Tre in the fusarium graminearum pH-1.
TABLE 4 accuracy and precision test results
Figure BDA0002984263080000091
The invention discloses a UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum, which selects an amino column and a T3 chromatographic column to be suitable for UPLC-MS/MS detection of two compounds of T6P and Tre, respectively establishes a method for simultaneously detecting T6P and Tre in fusarium graminearum by using the two chromatographic columns, and finds that the sensitivity of the amino column is high, the recovery rate of the T3 chromatographic column is high, and the stability is good. Two chromatographic columns are used for detecting that the content of T6P in fusarium graminearum pH-1 is respectively about 52.6 mug/g and 60.5 mug/g, and the content of Tre is about 4.42mg/g and 5.4 mg/g.

Claims (9)

1. A UPLC-MS/MS detection method of T6P and Tre in fusarium graminearum is characterized in that a sample solution is prepared after a sample fusarium graminearum is pretreated, the sample solution is eluted and separated through a chromatographic column, data are collected by using an MRM mode of UPLC-MS/MS, an electrospray ion source and monitoring in a negative ion scanning mode are carried out, an obtained spectrogram of a sample to be detected is compared with a standard spectrogram, corresponding substances are obtained when retention time is the same, and T6P and Tre are quantified respectively through a standard curve linear regression equation;
said chromatographic column selection
Figure FDA0002984263070000011
NH2
Figure FDA0002984263070000012
(100X 2mm, 3 μm) chromatography Column or a waters Acquity UPLC HSS T3 Column (100X 3mm, 1.7 μm) chromatography Column.
2. The UPLC-MS/MS detection method of T6P and Tre in Fusarium graminearum of claim 1, characterized in that: the pretreatment method of the fusarium graminearum sample to be detected comprises the following steps:
placing Fusarium graminearum to be detected in a mung bean soup liquid culture medium, culturing for 7 days at 28 ℃ and 140rpm, filtering to remove mycelia, centrifuging and concentrating to obtain 1 × 105Spore liquid per mL for later use; inoculating the spore liquid into a potato liquid culture medium, inoculating 1mL of the spore liquid into each bottle, performing shake culture at 28 ℃ and 140rpm for 7d, filtering to obtain hyphae, cleaning for 3 times by using deionized water, drying, and storing at-20 ℃ to be detected;
accurately weighing 0.04g of hypha sample in a 15mL centrifuge tube, adding 4mL deionized water, soaking for 1h, homogenizing by a high-speed homogenizer, adding 4mL methanol, freezing at-80 ℃, ultrasonically thawing and breaking the wall for 30min in an ultrasonic cleaner, centrifuging, and taking the supernatant to be tested.
3. The UPLC-MS/MS detection method of T6P and Tre in Fusarium graminearum according to claim 1, characterized in that the mass spectrometry conditions are specifically: an ion source: an electrospray ion source; the scanning mode is as follows: scanning negative ions; the detection mode is as follows: MRM multiple reaction monitoring Gas Curtain pressure (Curtain Gas:40 Kpa); electrospray Voltage (iosspray Voltage): -4500 Kpa; ion source Temperature (Temperature): at 450 ℃; atomization Gas pressure (Ion Source Gas 1): 65 Kpa; heat assist Gas pressure (Ion Source Gas 2): 65 Kpa; residence time: 40 ms.
4. The UPLC-MS/MS detection method of T6P and Tre in Fusarium graminearum of claim 1 or 3, characterized in that: when the detection object is T6P, the ion pair is: 421.2/241.1,421.2/139.1; DP was-110 and CE was-35, -35.
5. The UPLC-MS/MS detection method of T6P and Tre in Fusarium graminearum of claim 1 or 3, characterized in that: when the detection object is Tre, the ion pair is: 341.0/178.8,341.0/118.9; DP was-95 and CE was-19, -25.
6. The UPLC-MS/MS detection method of T6P and Tre in Fusarium graminearum of claim 1, characterized in that: the chromatographic column is
Figure FDA0002984263070000013
NH2
Figure FDA0002984263070000014
For a (100X 2mm, 3 μm) column, the UPLC optimization conditions were as follows:
mobile phase A: one ten thousandth of ammonia water;
mobile phase B: acetonitrile;
gradient: isocratic elution with 70% A;
flow rate: 0.4 mL/min.
7. The UPLC-MS/MS detection method of T6P and Tre in Fusarium graminearum of claim 1, characterized in that: when the chromatographic Column is a waters Acquity UPLC HSS T3 Column (100X 3mm, 1.7 μm), the UPLC optimization conditions are as follows:
mobile phase A: 5mM ammonium acetate (containing one ten thousandth of ammonia);
mobile phase B: acetonitrile;
gradient: 99% A at 0-3min, 10% A at 3-5min, and 99% A at 5-8 min;
flow rate: 0.25 mL/min.
8. The UPLC-MS/MS detection method of T6P and Tre in Fusarium graminearum of claim 1, characterized in that: the method for acquiring the T6P standard spectrogram specifically comprises the following steps:
weighing a T6P standard substance in a volumetric flask, dissolving the standard substance by using acetonitrile with volume fraction of 60%, fixing the volume, diluting the solution by using the acetonitrile with volume fraction of 60% to prepare standard working solutions with a plurality of concentration gradients step by step, carrying out UPLC-MS/MS analysis on the standard working solutions with different concentrations, and making a standard curve and a spectrogram.
9. The UPLC-MS/MS detection method of T6P and Tre in Fusarium graminearum of claim 1, characterized in that: the method for acquiring the Tre standard spectrogram specifically comprises the following steps:
weighing a Tre standard substance in a volumetric flask, dissolving the Tre standard substance by using acetonitrile with the volume fraction of 60% and fixing the volume, then diluting the Tre standard substance by using the acetonitrile with the volume fraction of 60% step by step to prepare a plurality of standard working solutions with concentration gradients, carrying out UPLC-MS/MS analysis on the standard working solutions with different concentrations, and making a standard curve and a spectrogram.
CN202110295681.9A 2021-03-19 2021-03-19 UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum Active CN113049705B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110295681.9A CN113049705B (en) 2021-03-19 2021-03-19 UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110295681.9A CN113049705B (en) 2021-03-19 2021-03-19 UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum

Publications (2)

Publication Number Publication Date
CN113049705A true CN113049705A (en) 2021-06-29
CN113049705B CN113049705B (en) 2023-04-07

Family

ID=76513793

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110295681.9A Active CN113049705B (en) 2021-03-19 2021-03-19 UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum

Country Status (1)

Country Link
CN (1) CN113049705B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106290606A (en) * 2016-07-25 2017-01-04 河南省科学院生物研究所有限责任公司 A kind of method of Quantitative detection trehalose synthase enzyme activity in antibacterial TreS approach trehalose producing strains screens
CN109358134A (en) * 2018-12-19 2019-02-19 武汉绿剑可瑞信科技有限公司 The pre-treating method and quantitative detecting method of endogenous sugar phosphate compound in a kind of plant sample
CN109900833A (en) * 2019-04-25 2019-06-18 中国农业科学院蔬菜花卉研究所 The ultra performance liquid chromatography tandem mass spectrum detection method of trehalose-6-phosphate content in tomato

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106290606A (en) * 2016-07-25 2017-01-04 河南省科学院生物研究所有限责任公司 A kind of method of Quantitative detection trehalose synthase enzyme activity in antibacterial TreS approach trehalose producing strains screens
CN109358134A (en) * 2018-12-19 2019-02-19 武汉绿剑可瑞信科技有限公司 The pre-treating method and quantitative detecting method of endogenous sugar phosphate compound in a kind of plant sample
CN109900833A (en) * 2019-04-25 2019-06-18 中国农业科学院蔬菜花卉研究所 The ultra performance liquid chromatography tandem mass spectrum detection method of trehalose-6-phosphate content in tomato

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JAVIER SASTRE TORANO ET AL.: "Determination of trehalose-6-phosphate in Arabidopsis thaliana seedlings by hydrophilic-interaction liquid chromatography-mass spectrometry", 《ANAL BIOANAL CHEM》 *
何秀全等: "HPLC-ELSD法测定木薯叶片中海藻糖的含量", 《热带作物学报》 *
张曰辉等: "高效液相色谱法检测奶糖中海藻糖等糖含量的研究", 《中国食品添加剂》 *
董玲玲等: "液质联用方法测定白色念珠菌中海藻糖的含量", 《药学实践杂志》 *

Also Published As

Publication number Publication date
CN113049705B (en) 2023-04-07

Similar Documents

Publication Publication Date Title
CN111912926A (en) Method for determining reduced glutathione content in rice by ultra-high performance liquid chromatography-tandem mass spectrometry
CN109030658B (en) Method for detecting fructo-oligosaccharide and raffinose in infant milk powder
CN113390992A (en) Method for analyzing and detecting trace aflatoxin in water-soluble fermented medicine
CN112666273A (en) Method for detecting concentration of methotrexate substances in erythrocytes
CN105842377B (en) The high-efficiency liquid chromatography method for detecting of pyrazine compounds in a kind of white wine
Chen et al. Determination of ellagic acid in wine by solid-phase extraction–ultra-high performance liquid chromatography–tandem mass spectrometry
CN108508130B (en) Method for splitting and measuring enantiomers of chiral pesticides metalaxyl and dimethomorph by ultra-high performance combined chromatography-tandem mass spectrometry technology
CN109212042B (en) Analysis method for determining toxicity impurities of pezopyr hydrochloride gene by adopting liquid chromatography-mass spectrometry
CN110133169A (en) A kind of method and application using frusemide in LC-MS detection human plasma
CN111983055B (en) Method for separating and measuring rivaroxaban intermediate related substances by using HPLC (high performance liquid chromatography)
CN113049705B (en) UPLC-MS/MS detection method for T6P and Tre in fusarium graminearum
CN108072717B (en) Method for detecting arginine solution
CN113917013B (en) Method for measuring methanol and 5 higher alcohols in fruit distilled liquor by internal standard method
CN111239303A (en) Method for simultaneously determining concentrations of ticagrelor, active metabolites thereof and endogenous adenosine in human plasma by liquid chromatography-mass spectrometry
CN108663464B (en) Method for detecting fenhexamid in fruits, vegetables or soil
Yu et al. Determination of myriocin in natural and cultured Cordyceps cicadae using 9-fluorenylmethyl chloroformate derivatization and high-performance liquid chromatography with UV-detection
CN108132309A (en) The analysis method of Thiodiazole-copper residual quantity in a kind of measure dendrobium candidum
CN110836930A (en) Method for measuring content of dichlorobutane in levetiracetam by gas chromatography-mass spectrometry
CN114034799A (en) Method for detecting fluxapyroxad residual quantity in grapes
CN114137120A (en) Method for detecting related substances in rapamycin drug stent
CN111521693B (en) Method for detecting isosorbide mononitrate
CN112114080B (en) Method for detecting aripiprazole related substances
CN112461975A (en) Method for detecting coenzyme content in feed additive
CN111751466A (en) Method for simultaneously determining DON toxin and NIV toxin
CN111610275A (en) Method for determining malto-oligosaccharide and application of method in field of product quality control

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant