Jan 21, 2025
LC-MS analysis for plant global metabolomics: C18 analysis.
This protocol is a draft, published without a DOI.
- Sylvie Fournier1,2,
- Amélie Perez1,2
- 1CNRS;
- 2Metatoul-Agromix
- MetaboHUB-MetaToul
External link: https://lrsv.cnrs.fr/metatoul-en/
Protocol Citation: Sylvie Fournier, Amélie Perez 2025. LC-MS analysis for plant global metabolomics: C18 analysis.. protocols.io https://protocols.io/view/lc-ms-analysis-for-plant-global-metabolomics-c18-a-de9n3h5e
Manuscript citation:
Justine Chervin, Ana Romeo-Oliván, Sylvie Fournier, Virginie Puech-Pagès, Bernard Dumas, et al.. Modification of Early Response of Vitis vinifera to Pathogens Relating to Esca Disease and Biocontrol Agent Vintec‱ Revealed By Untargeted Metabolomics on Woody Tissues. Frontiers in Microbiology, 2022, 13, pp.835463. ⟨10.3389/fmicb.2022.835463⟩. ⟨hal-03652523⟩
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 07, 2024
Last Modified: January 21, 2025
Protocol Integer ID: 101390
Keywords: LC-MS global analysis, C18, plant
Funders Acknowledgements:
Agence Nationale de la Recherche
Grant ID: ANR-11-INBS-0010 MetaboHUB
Abstract
The LC-MS analysis for global metabolomics was carried out on a Thermo Scientific Vanquish UHPLC chromatography and Thermo Scientific Q Exactive Plus mass spectrometer. The Column was a Luna omega Polar C18 (Phenomenex 1.6 µm, 150 × 2.1 mm). Injection temperature and volume were 10° C and 5 µL for final sample concentration at 1 mg/ml. Elution parameters consisted of a flow rate of 0.4 mL/min with water acidified (0.05% formic acid) as solvent A and acetonitrile acidified (0.05% formic acid) as solvent B using the following gradient: 2% B (0 – 0.5 min), 2 to 70% B (0.5 - 8 min), 70 to 98% B (8 - 9 min) 98% B (9 - 12 min), 98 to 2% B (12 – 12.1 min) and 98%B (12.1 – 14 min). DAD and CAD detectors can be used.
Image Attribution
Sylvie Fournier,
Amélie Pérez
Materials
Equipment
U-HPLC Vanquish - Q Exactive Plus
NAME
U-HPLC-HRMS
TYPE
Thermo Scientific
BRAND
Q-Exactive Plus + Vanquish double pump, CAD, DAD
SKU
Software
Thermo Scientific Xcalibur
NAME
Equipment
Glass vial 1.5ml
NAME
Glass vial
TYPE
Fisherbrand
BRAND
11575884
SKU
9 mm Short Thread Glass Vial, Wide Opening
SPECIFICATIONS
Equipment
Verex Insert, 175µL
NAME
Insert
TYPE
Phenomenex
BRAND
AR0-4521-13
SKU
Verex Insert, 5mm Dia., 175µL, Clear 51, Conical Bottom, w/ bottom spring
SPECIFICATIONS
Equipment
Preslit Screw cap
NAME
Screw cap
TYPE
Fisherbrand
BRAND
11797567
SKU
9mm PP Short Thread Seal, Blue, Center Hole, Assembled Septum
SPECIFICATIONS
Equipment
C18 Polar LC Column, 150 x 2.1 mm
NAME
LC column
TYPE
Luna Omega
BRAND
00F-4748-AN
SKU
LINK
Luna Omega 1.6µm Polar C18 100Å, LC Column 150 x 2.1 mm, EA
SPECIFICATIONS
Equipment
SecurityGuard ULTRA holder
NAME
Guard column holder
TYPE
Phenomenex
BRAND
AJ0-9000
SKU
LINK
2.1 to 4.6mm ID
SPECIFICATIONS
Protocol materials
Ultrapure WaterMilli-Q
Isopropanol, Certified AR for Analysis, Fisher ChemicalFisher ScientificCatalog # 10366430
Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
Acetonitrile LC-MS GradeFisher ScientificCatalog #A/0638/15
Safety warnings
Wear appropriate clothing, closed shoes, lab coat, glasses, gloves, extractor hood
Use the most recent version of the Instrument methods:
folder : Data (D)> Acquisition> METHODS-LC-MS>Metabo
Criterion of acceptability of results:
| A | B | |
| Criteria | Conformity | |
| Controled and maintened instruments | ok | |
| Pressure variability between the first and the last samples | < 80 bars | |
| Inter-analysis RT difference | < 0.5 min | |
| Intra-analysis RT difference | < 0.2 min | |
| Memory effect | No detectionin blank (S/N<3) | |
| PCA (not for fingerprinting) | Centered QC | |
| Monthly injection of a standard mix | No variation in chromatograms between 2 months |
Before start
If necessary, clean the Ionization source (cf instruction nettoyage)
Calibrate the mass spectrometer (cf. Instruction calibration)
Equilibrate the column
Injection at least of 6 blanks before the first analysis.
Ensure that samples are correctly identified to ensure traceability.
Sample type
Sample type
Extracts of plant and associate microorganism
1 mg/mL extract sample
Quality control
Quality control
Prepare a Quality Control (QC) = mix of all samples, a blank (solvent) and a blank of extraction.
Reagents and Equipments
Reagents and Equipments
Reagents for mobiles phase and LC system
Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260 Acetonitrile LC-MS GradeFisher ScientificCatalog #A/0638/15
Ultrapure WaterMilli-Q
Isopropanol, Certified AR for Analysis, Fisher ChemicalFisher ScientificCatalog # 10366430
Equipments
Equipment
SecurityGuard ULTRA holder
NAME
Guard column holder
TYPE
Phenomenex
BRAND
AJ0-9000
SKU
LINK
2.1 to 4.6mm ID
SPECIFICATIONS
Equipment
C18 Polar LC Column, 150 x 2.1 mm
NAME
LC column
TYPE
Luna Omega
BRAND
00F-4748-AN
SKU
LINK
Luna Omega 1.6µm Polar C18 100Å, LC Column 150 x 2.1 mm, EA
SPECIFICATIONS
Equipment
Verex Insert, 175µL
NAME
Insert
TYPE
Phenomenex
BRAND
AR0-4521-13
SKU
Verex Insert, 5mm Dia., 175µL, Clear 51, Conical Bottom, w/ bottom spring
SPECIFICATIONS
Equipment
Preslit Screw cap
NAME
Screw cap
TYPE
Fisherbrand
BRAND
11797567
SKU
9mm PP Short Thread Seal, Blue, Center Hole, Assembled Septum
SPECIFICATIONS
Equipment
Glass vial 1.5ml
NAME
Glass vial
TYPE
Fisherbrand
BRAND
11575884
SKU
9 mm Short Thread Glass Vial, Wide Opening
SPECIFICATIONS
Equipment
U-HPLC Vanquish - Q Exactive Plus
NAME
U-HPLC-HRMS
TYPE
Thermo Scientific
BRAND
Q-Exactive Plus + Vanquish double pump, CAD, DAD
SKU
Software
Thermo Scientific Xcalibur
NAME
LC-MS Preparation
LC-MS Preparation
Analysis are done on a Q-exactive Plus (ThermoScientific) spectrometer equipped with a heated
electrospray (HESI II) or APCI probe, a Diode Array Detector (DAD, ThermoScientific), a Charged Aerosol Detector (CAD, ThermoScientific) and coupled with U-HPLC system (Vanquish model H,ThermoScientific).
Equipment
U-HPLC Vanquish - Q Exactive Plus
NAME
U-HPLC-HRMS
TYPE
Thermo Scientific
BRAND
Q-Exactive Plus + Vanquish double pump, CAD, DAD
SKU
Column: Phenomenex Luna omega Polar C18 1.6 µm, 150 × 2.1 mm + UHPLC Fully Porous Polar C18 2.1mm precolumn (SecurityGuard ULTRA Cartridges, Phenomenex)
Equipment
C18 Polar LC Column, 150 x 2.1 mm
NAME
LC column
TYPE
Luna Omega
BRAND
00F-4748-AN
SKU
LINK
Luna Omega 1.6µm Polar C18 100Å, LC Column 150 x 2.1 mm, EA
SPECIFICATIONS
Equipment
SecurityGuard ULTRA Holder
NAME
guard column holder
TYPE
Phenomenex
BRAND
AJ0-9000
SKU
LINK
2.1-4.6 mm ID
SPECIFICATIONS
Mobile phase for gradient:
A1: H2O + 0.05% Formic acid
B1: ACN + 0.05% Formic acid
Mobile phase for CAD counter gradient:
A: ACN + 0.05% Formic acid
B: H2O + 0.05% Formic acid
Mobile phase for injection loop: ACN/H2O (50/50)
Mobile phase for pump head 1: IPA/H2O (75/25) + 0.05% Formic acid
Mobile phase for pump head 2: H20/IPA (90/10)
Elution gradient
(same for Pump 1 (column) and Pump 2 (CAD)) :
| Time (min) | % A1 (pump 1) % B (pump 2 CAD) | % B1 (pump 1) % A (pump 2 CAD) | |
| 0 | 98 | 2 | |
| 0.5 | 98 | 2 | |
| 08 | 30 | 70 | |
| 09 | 2 | 98 | |
| 12.0 | 2 | 98 | |
| 12.1 | 98 | 2 | |
| 14.0 | 98 | 2 |
DAD conditions:
Three usual UV-vis wavelenghts are programmed:
* UV-vis 1 = 254 nm
* UV-vis 2 = 200 nm
* Uv-vis 3 = 320 nm
CAD conditions:
Split is adjusted to 75
Injection Volume:
5 µl (@ 1mg/ml)
LC-MS Instrument method
LC-MS Instrument method
Acquisitions are done in ESI full scan + MS/MS in positive and negative modes.
Location of Instrument methods: folder Data (D)> Acquisition> METHODS-LC-MS>Metabo
Copy in your folder, the most recent version
C18 LC parameters (January 2025):
Pump 1 General settings
Pump 1 flow gradient
Pump 2 General settings
Pump 2 flow gradient
Sampler module
Sampler module, temperature control
Column compartiment (Preheat: Off)
UV settings
UV settings
CAD settings
CAD settings
System
Smart Startup
Smart Shutdown
Smart Standby
C18 MS parameters (January 2025):
Global list - only exclusion list used
Tune File
External Hardware - Only divert valve a used
MS experiment : Full MS/ddMS2
MS experiment : Full MS/ddMS2 parameters
Sequence
Sequence
Prepare a Quality Control (QC) = mix of all samples, a blank (solvent) and a blank of extraction.
Inject QC and blank (solvent) every 6 injections.
Example of a typical profile of standards
Example of a typical profile of standards
In this example, a standard mix is injected at 10 µg/mL with the parameters described before in positive and negative mode.
Chromatogram of the standard mix in positive mode (5µL)
: Chromatogram of the standard mix in negative mode (5µL)
Data processing
Data processing
cf tutorial data processing Agromix
Status of updates
Status of updates
| Version | Date created | Nature of changes | |
| 01 | 21/05/25 | Document creation on protocols.io | |
| 02 | |||
| 03 |