Jan 22, 2025
Version 1
Extraction of polar metabolites and isotope profiling using HPLC-HRMS V.1
This protocol is a draft, published without a DOI.
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Protocol Citation: Léana Porcher-Bibes 2025. Extraction of polar metabolites and isotope profiling using HPLC-HRMS. protocols.io https://protocols.io/view/extraction-of-polar-metabolites-and-isotope-profil-de7j3hkn
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 06, 2024
Last Modified: January 22, 2025
Protocol Integer ID: 101323
Keywords: Liquid Chromatography, High-Resolution, High-Resolution Mass Spectrometry, Polar metabolites, Extraction, Isotope analysis, Fluxomics, Tracing
Abstract
The extraction of polar metabolites from biological samples and the analysis of 13C incorporation into metabolites via HPLC-HRMS using the Thermo Q-Exactive Plus instrument are described.
Sample preparation is first presented, followed by tissue and biofluid polar metabolites extration, and finally the analytical parameters are described.
Analysis of the incorporation of (stables) isotopes into metabolites in the context of labelling experiments carried out on animals or Human, to measure the fate of the labelled compounds and the dynamics of metabolism (fluxomics)
Materials
· Blouse
· Gloves
· Goggles
· Suitable chemical hood and BOA (Orientable Aspirating Arm)
· Freezer -80°C
· Freezer -20°C
· Ice
· Freeze-dryer
· Vibratory shredder
· 5mm grinding balls
· miVac Quattro evaporator
· Centrifuge 4°C
· Eppendorf racks
· 1.5ml Eppendorf Safe Lock tubes
· 2ml glass vials
· 250µl glass inserts
· P1000+cones
· P100+cones
· P10+cones
· 19G needle
· Small spatula
· Vortex
· Ultrapure water
Protocol materials
Méthanol, LC-MS CHROMASOLV™Honeywell FlukaCatalog #15624740
Acetonitrile hypergradeFisher ScientificCatalog #10616653
Ammonium acetate, 98%Fisher ScientificCatalog #10543961
Acetonitrile hypergradeFisher ScientificCatalog #10616653
Ammonium acetate, 98%Fisher ScientificCatalog #10543961
Acetonitrile hypergradeFisher ScientificCatalog #10616653
Ammonium acetate, 98%Fisher ScientificCatalog #10543961
Méthanol, LC-MS CHROMASOLV™Honeywell FlukaCatalog #15624740
Acetonitrile hypergradeFisher ScientificCatalog #10616653
Formic Acid, 98%Fisher ScientificCatalog #10071620
Safety warnings
This protocol involves the use of volatile solvents that may pose health risks. Therefore, it is essential to handle these solvents within a designated chemical fume hood and to wear appropriate personal protective equipment (gloves, goggles, lab coat, etc.).
Always handle samples on ice and maintain a cold environment to prevent degradation of the metabolites of interest.
Frozen tissues should be stored at -80°C in 2 mL Eppendorf Safe-Lock tubes with a round bottom, except for liver tissue, which should be stored in 5 mL Eppendorf Safe-Lock tubes.
Before start
Before starting, prepare the solution suitable for the intended extraction :
For plasma extraction prepare 1L of 80/20, ACN/H2O
For tissues extraction prepare 1L of MeOH/ACN/H2O, 2/2/1 +125mM formic acid
Verify the availability of the internal standard.
Ensure that tubes, beads, and extraction supports are stored in the freezer (-20°C).
Confirm that samples are correctly labeled for traceability.
Sample preparation
Sample preparation
Sample preparation
This section outlines the preparation of biological samples (plasma, kidney, liver, muscle, lungs...) and the extraction of polar metabolites. The process is divided into four main stages:
- Sample preparation: freeze-drying, grinding, and weighing of tissues
- Extraction of polar metabolites from tissues
- Extraction of polar metabolites from biofluids
- Tissues and biofluids sample recovery
Tissue freeze-drying
- Weigh frozen tissues with their tubes before freeze-drying
- Pierce 3 holes in the cap of tubes containing frozen tissue using a 19G syringe needle.
Equipment
19G Syringe Needle
NAME
Dutscher
BRAND
050122B
SKU
- Place frozen tissues with their pierced tubes in the freeze-dryer
- Freeze-dry for 36 hours
- Weigh freeze-dried tissues with their pierced tubes after freeze-drying
- Replace pierced caps with new, undrilled caps
- Store at -80°C after parafilming the cap or continue with grinding
Grinding & weighing
Grinding :
- Place cold stainless steel beads 5mm in tubes containing freeze-dried tissue
Equipment
Stainless steel beads 5 mm
NAME
Qiagen
BRAND
69989
SKU
- Grind with a vibro-grinder for 30s at 30Hz cold several times to obtain a powder, (maintain the blocks on a dry ice bed between each run and return them to -20°C at the end of the procedure)
Equipment
Vibro Grinder
NAME
Retsch
BRAND
MM 400
SKU
Weighing:
- Keep the tubes containing lyophilized/ground tissue in the dry ice.
- Weigh 5mg of lyophilized/ground tissue into 2ml Eppendorf Safe Lock round-bottom tubes
- Store at -80°C until the extraction
Extraction of polar metabolites from tissues
The solution used for this extraction is a mixture of MeOH/ACN/H2O, 2/2/1 +125mM formic acid
Méthanol, LC-MS CHROMASOLV™Honeywell FlukaCatalog #15624740
Acetonitrile hypergradeFisher ScientificCatalog #10616653
Formic Acid, 98%Fisher ScientificCatalog #10071620
- Withdraw and transfer a sufficient amount of solution into another container to avoid contaminating the stock solution
- Add to the eppendorf containing 5 mg of ground tissue : 1.5 mL of cold extraction solution (MeOH/ACN/H2O, 2/2/1 +125mM formic acid - shake well and degas the solution before use) and one cold 5mm stainless steel beads
Equipment
Stainless Steel 5 mm beads
NAME
Qiagen
BRAND
69989
SKU
- Grind 3 times for 30s at 30Hz cold (keep the blocks on the dry ice bed between each run and return the blocks to -20°C at the end)
- Incubate for 15 min at -20°C (upside-down incubation for adipose tissue)
- Vortex 1 min
- Collect 50µl for protein assay after vortexing each tube (store at -20°C)
- Centrifuge 16000 x g, 4°C, 00:15:00
- Collect 1ml of supernatant and store in a 2ml Eppendorf Safe Lock round-bottom tube (keep the tubes on the dry ice bed)
- Add 500µl of cold extraction solution to the pellet (/!\ shake well and degas the solution before use)
- Grind 2 times for 30s at 30Hz cold (keep the blocks on the dry ice bed between each run)
- Incubate for 10 min at -20°C (upside-down incubation for adipose tissue)
- Vortex 1 min
- Centrifuge 16000 x g, 4°C, 00:15:00
- Collect 600µl of supernatant and add to 1ml already collected (keep the tubes on the dry ice bed)
- Evaporate collected supernatant with miVac Quattro at room temperature using the H2O method with freezing mode (check evaporation each four hours)
if evaporation is not possible immediately, samples can be stored at -80°C
- Store evaporated samples at -80°C
Extraction of polar metabolites from biofluids
The solution used for this extraction is a mixture of MeOH/H2O, 80/20
Méthanol, LC-MS CHROMASOLV™Honeywell FlukaCatalog #15624740
Withdraw and transfer a sufficient amount of solution into another container to avoid contaminating the stock solution
- Take 20 µL of plasma or an adapted volum of biofluid and add 1 mL of cold extraction solution (MeOH/H2O, 80/20 - shake well and degas solution before use)
- Vortex 00:01:00
- Perform 3 cycles : incubate for 00:05:00 at -20 °C + vortex 00:01:00
- Sonication 00:03:00 add ice to ultrasonic bath
- Vortex 00:01:00
- Collect 50 µL of supernatant for protein assay after vortexing each tube (store at -20 °C )
- Centrifuge sample epperdorf at 16000 x g, 4°C, 00:15:00
- Collect 850 µL of supernatant
- Evaporate all supernatant with miVac Quattro at room temperature using the H2O method with freezing mode (caps open and hinges facing outwards, check evaporation each four hours)
(if evaporation is not possible immediately, samples can be stored at -80 °C ).
- Store evaporated samples at -80 °C
Tissues and biofluids sample recovery before LC-HRMS analysis
The solution used for this sample recovery is a mixture of ACN/H2O, 3/1 +15 mM ammonium acetate
Acetonitrile hypergradeFisher ScientificCatalog #10616653
Ammonium acetate, 98%Fisher ScientificCatalog #10543961
- Add an adapted volume of the sample recovery solution to the evaporated samples
- Vortex 00:01:00
- Perform 3 cycles: incubate 00:05:00 at -20 °C then vortex 00:01:00
- Centrifuge 16000 x g, 4°C, 00:15:00
- Transfer 35 µL to a vial with insert
Preparation of the Quality Control : depending on the number of samples, pool 3 to 10µl of each sample in a vial with insert for Quality Control (vortex each sample before pooling)
Isotope profiling analysis using HPLC-HRMS
Isotope profiling analysis using HPLC-HRMS
Analysis of polar metabolites by HPLC-HRMS
This section describes the analytical conditions for the analysis of polar metabolites after their extraction from biological sample.
Analysis are done on a HPLC system (Vanquish,ThermoScientific) coupled to a Q-exactive Plus (ThermoScientific) spectrometer equipped with a heated electrospray (HESI)
Equipment
HPLC Vanquish
NAME
Liquid chromatography
TYPE
Thermo Scientific
BRAND
qqq
SKU
Coupled to a QExactive plus High Resolution Mass Spectrometer
SPECIFICATIONS
This section is divided into 2 main stages:
1. HPLC parameters
2. HRMS parameters
HPLC parameters
- Column :
Equipment
P120 HILIC-Z, 2,1×150 mm, 2,7 µm
NAME
HPLC Column
TYPE
Agilent Technologies
BRAND
673775-924
SKU
- Guard column :
Equipment
Guard Poroshell 120 5x2.1, 2.7 µm
NAME
Guard column
TYPE
Agilent Technologies
BRAND
821725-947
SKU
- Mobile phase :
Mobile phase A : 90/10, ACN/H2OmQ + 15 mM ammonium acetate
Acetonitrile hypergradeFisher ScientificCatalog #10616653
Ammonium acetate, 98%Fisher ScientificCatalog #10543961
Mobile phase B : 10/90 ACN//H2OmQ + 15 mM ammonium acetate
Acetonitrile hypergradeFisher ScientificCatalog #10616653
Ammonium acetate, 98%Fisher ScientificCatalog #10543961
- Flow : 0.250 mL/min
- T° Sampler : 4°C
- Column T° : 30°C
- Injection volume : 2-5 µL
- Gradient :
HPLC Flow gradient 1/2
HPLC flow gradient 2/2
HRMS parameters
| A | B | |
| Tune | 2700_300_tune | |
| Source | HESI | |
| Mode | Negative | |
| Acquisition | Fullscan | |
| Résolution | 70 000 | |
| T° Capillaire | 250 | |
| Sheath gas flow rate | 45 | |
| Auxiliary gas flow rate | 10 | |
| S-Lens RF level | 60 | |
| Tension source | 2,70 kV | |
| Sweep Gas flow rate | 1 |
Verification of the analytical system
Verification of the analytical system
Verification of the analytical system
This section outlines the steps to verify the system prior to sample injection to ensure proper functionality
Orbitrap mass spectrometer calibration
Before analysis, calibrate the mass spectrometer with the adapted solution
Pre/post-extraction solvent analysis
The sequence should begin with the injection of a “pre-extraction” and “post-extraction” solvent. These injections confirm that no contamination has occurred during the sample extraction process.
Quality Control
A quality control should be injected throughout the sequence every 6 to 10 injections to verify the analytical conditions.
Blank
A blank should be injected throughout the sequence every 6 to 10 injections to confirm that no contamination has occurred during the sample extraction process
PA-TP (isotopic analysis)
A “PA-PT” sample (Pascal's triangle sample of proteinogenic amino acids) is injected at the end of the analytical batch to check the accuracy of the mass spectrometer for isotopic analysis.
3 amino acids should be integrated (Aspartate, Arginine and Phenylalanine) and the bias obtained on the CID (measured values vs. theoretical values) must be less than 5%. In the case of large analytical sequences (> 30 samples), the TP sample should be injected at the end of each series of 30 samples.
Sample management and storage
Sample management and storage
Sample management and storage
This section describes how samples are treated after analysis
After analysis, the injection vials are discarded.
Remaining samples in the eppendorf are stored at -80°C for up to 5 years. It is also possible to return them to the requesting laboratory, at the latter's expense.