Jan 30, 2026
Version 2
Biphasic extraction metabolites in algal matrix or periphyton V.2
Forked from a private protocol
- Mélissa EON1,
- Nicolas Creusot1
- 1INRAE
- MetaboHUB-Bordeaux
- MetaboHUB
Protocol Citation: Mélissa EON, Nicolas Creusot 2026. Biphasic extraction metabolites in algal matrix or periphyton. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1zxdlmk/v2Version created by Mélissa ME EON
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: January 06, 2026
Last Modified: January 30, 2026
Protocol Integer ID: 237133
Keywords: metabolites, environnemental metabolomics, biphasic extraction, algal matrix, periphyton, lipids on an algal matrix, biphasic extraction, different steps of extraction, h2o mixture for the simultaneous extraction, liquid extraction, lipid metabolite, metabolite, extraction, simultaneous extraction, algal matrix, lipid, periphyton the purpose, periphyton, h2o mixture
Funders Acknowledgements:
MetaboHUB
Grant ID: ANR-11-INBS-0010
ANR-21-ESRE-0035
Grant ID: ANR-21-ESRE-0035
Abstract
The purpose of this instruction is to specify the different steps of extraction of metabolites and lipids on an algal matrix or periphyton previously freeze-dried and stored in a freezer at -80 °C .
Principle:
Use of a MeOH/MTBE/H2O mixture for the simultaneous extraction of primary, secondary and lipid metabolites. Extraction is done by combining liquid/solid and liquid/liquid extraction that is carried out twice to optimize yields.
Materials
Consumable
Benchtop with extractor hood
2 mL tubes with fl at cap skirt in PP + 0.5mm microbeads
Ice blocks and ice cubes
Micropipettes + tips
Amber Vials 1.8mL with cryopreservation label
150mL glass bottles
UPLC grade solvents
2 beakers
Internal standards and matrix for control
100 mg of spike matrix (control condition or reference matrix)
PE tracer solution (15:0) @ 100 ng.μL-1 (or ppm)
Internal "polar" lipid standards @ 33.3 ng.μL-1 (or ppm)
Mix SST (50 mg/L)
Pesticide mix (3.3 mg/L)
Mix IS pesticides (2.5 mg/L)
Mix IS metabolites (50 mg/L) + (100mg/L)
Solvents
Ultra pure water :H2O
ACETONITRILE :ACN
METHANOL : MEOH
ISOPROPANOL : ISO
Equipment
Ultrasonic cleaner USC100T
NAME
Ultrasonic tank
TYPE
VWR
BRAND
6
SKU
Equipment
Labconco™ FreeZone™ 2.5L -84°C Benchtop Freeze Dryer
NAME
FreeZone
BRAND
10-400-014
SKU
LINK
Precision scale
Equipment
scale SARTORIUS
NAME
scale
TYPE
SARTORIUS
BRAND
MSA 36P-000DH
SKU
Equipment
FastPrep-24 5G
NAME
Bead beater
TYPE
MP Biomedicals
BRAND
116005500
SKU
LINK
Equipment
5430R
NAME
refrigerate centrifuge
TYPE
Eppendorf
BRAND
5430 R
SKU
Equipment
new equipment
NAME
Genevac EZ-2
BRAND
EZ-2
SKU
https://www.spscientific.com/Products/Centrifugal_Evaporators___Sample_Concentrators/Genevac/EZ-2_Series/EZ-2_Series/
SPECIFICATIONS
Protocol materials
MTBE: Sigma Chromasolv 99.8% for HPLC 100mL (smallest available) (34875-100mL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34875-100ML
MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641
MilliQ water
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
Isopropanol HyperSolv CHROMANORMVWR International (Avantor)
Troubleshooting
Safety warnings
Use lab coat, gloves and safety glasses and laboratory fume hood. You can refere to the safety rules of the lab.
All waste generated must be treated properly.
Sample traceability is paramount, so it is necessary to optimize sample naming and storage as well as all reagents and stock solutions.
Gravimetric monitoring of stock solutions is carried out.
Write down all the weight as well as the volumes and nature of the internal standards added in a laboratory notebook.
Before start
Samples must be freeze dried and store at -80°C
Cleaning of the glassware should be done according to the laboratory laundry protocol using UPLC grade solvents or 8h oven calcination at 450°C.
All reagents used, including water, must be UPLC grade.
As the preservation of samples is done by cold, it is essential that as many steps as possible are done on ice.
EXTRACTION OVERVIEW
Extraction overview
PREPARATION
2h
EXPERIMENTAL CONDITIONS
Allow 4-6 replicates per experimental sample
o For environmental samples: provide doping with pesticide internal standards (IS) + metabo/lipido IS
o For laboratory samples: provide doping with IS of the investigated compound + metabo/lipido IS
• Provide a Procedural blank = solvent condition (including or not white field) => 3 replicates
• Provide a condition Blind Blank = solvent (=procedural blank with all IS) => 2 replicates
• Provide Spikes for validation of the manipulation => 2 replicates
o Spike (1) reference sample + (SST + Pest + PE:15) pre Extraction. + IS post Extraction
o Spike (2) reference sample + IS post Extraction.
PREPARATION OF EXTRACTION MIXTURES – UPLC GRADE (HERE FOR 60 REPLICATES!)
• Prepare an MTBE:MeOH mixture (3:1, v:v) =>150 mL stored at 4 °C for maximum one week
• Prepare a mixture H2O:MeOH (3:1, v:v) => 100 mL stored at 4 °C for maximum one week
MTBE: Sigma Chromasolv 99.8% for HPLC 100mL (smallest available) (34875-100mL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34875-100ML MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641
MilliQ water
PREPARATION OF BALL TUBES AND VIALS
All these steps can be done the day before.
• Annotate all tubes
• Add 150 mg (± 10mg) of 0.5mm beads to each 2mL tube
• Annotate all 1.8mL amber stock vials (duplicate for lipophilic and hydrophilic fractions)
PREPARATION OF CRUSHED ICE OR COLD BLOCKS
• Prepare ice with the ice machine continuously throughout the extraction
• Place cold blocks at -80 °C as well as microtube racks
PREPARATION OF MIXTURES FOR DOPING:
SST mix (50 Parts per Million (PPM) ) + pesticide mix (3.3 Parts per Million (PPM) )mix IS pesticides (2.5 Parts per Million (PPM) ) + mix IS metabo (50 Parts per Million (PPM) ) + PE (15:0) (100 Parts per Million (PPM) )
• Ultrasound 00:05:00 at Room temperature
5m
EXTRACTION STEPS
2h
PRELIMINARY STEPS
On ice, in the soundcheck room:
• Add 10 mg (+- 1mg) of freeze dried matrix per tube containing the microbeads
• Record the exact mass of sample added
Doping with tracers/surrogates and IS:
o Sample approx: 10 µL IS pesticides + 10 µL IS metabo + 50 µL PE (15:0)
o Lab sample: 10 µL IS of the test compound + 10 µL IS metabo + 10 µL PE (15:0)
o Spike on matrix (or solvent if matrix not available): Extraction yield
Spike 1:
• Pre-extraction: 10 µL mix SST + 10 µL mix Pest + PE (15:0)
o Procedural blank: solvent only
o Blind blank: solvent + 10 µL All IS
In order to guarantee the cold chain:
• For 00:15:00 : Place the tubes and MTBE:MeOH solution at -80 °C ; H20:MeOH solutions at -20 °C
• Start the Fast Temp on the centrifuge 4 °C
15m
EXTRACTION 1
Under hood and on ice: Add 1 mL of MTBE:MeOH (3:1) per tube
• Cycle to Fast prep:00:00:15 at 6.5 m/s
• Place the samples in ice or at -80 °C for 00:03:00
• Do a second cycle in Fast prep: 00:00:15 at 6.5 m/s
• Place the samples in ice or at -80 °C for 00:03:00
Under hood and on ice: Add 650 µL of H2O:MeOH
• Do a third cycle in Fast prep: 00:00:15 at 6.5 m/s
• Place the samples in ice or at -80 °C for 00:03:00
• Centrifugation: 12000 rpm, 4°C, 00:05:00
Under the hood and on ice, recover the phases in vials stocks (amber 1.8mL)
=>500 µL lipophilic above; 600 µL hydrophilic below using a micropipette
Save the base for extraction 2
Equipment
FastPrep-24 5G
NAME
Bead beater
TYPE
MP Biomedicals
BRAND
116005500
SKU
LINK
14m 45s
EXTRACTION 2
Under hood and on ice: Add 700 µL of MTBE:MeOH (3:1) per tube
• Cycle to Fast prep:00:00:15 at 6.5 m/s
• Place the samples in ice or at -80 °C for 00:03:00
• Do a second cycle in Fast prep: 00:00:15 at 6.5 m/s
• Place the samples in ice or at -80 °C for 00:03:00
Under hood and on ice: Add 455 µL of H2O:MeOH
Do a third cycle in Fast prep: 00:00:15 at 6.5 m/s
• Place the samples in ice or at -80 °C for 00:03:00
Centrifugation: 12000 rpm, 4°C, 00:05:00
Under the hood and on ice, recover the phases under the hood in the same vials
=>600 µL lipophilic above;700 µL hydrophilic below using a micropipette
End of the process
• Weigh the amber stocks after transfer of the extracts
=>Theoretically: 1.1 mL lipo vs 1.3 mL hydro
•Doping
o Spike 1 & 2
• Post-extraction:10 µL IS metabo + 10 µL IS pesticides
• Store all vials at-80 °C
14m 45s
PREPARATION OF VIALS FOR INJECTION
2h
HYDROPHILIC FRACTION UPLC-TOF
• 00:05:00 ultrasound
•500 µL transfer into clear vials
• Evaporate solvant in Genevac : 30 °C , 00:30:00 hydro mode
• Add 100 µL H2O:ACN 50:50 and transfer into insert
Acetonitrile ULC-MSBiosolveCatalog #0001204101BS
• QC pool preparation and dilutions
=>Pool 10 µL of each sample in amber vial (without insert) and prepare QC/2 QC/4
Boosting the QC pool with leucine 1ppm at 1/100
• Calibration range preparation: SST mix + Pest mix + PE (15:0) with fixed concentration IS metabo and Pest
Equipment
new equipment
NAME
Genevac EZ-2
BRAND
EZ-2
SKU
https://www.spscientific.com/Products/Centrifugal_Evaporators___Sample_Concentrators/Genevac/EZ-2_Series/EZ-2_Series/
SPECIFICATIONS
35m
LIPOPHILIC FRACTION UPLC-TOF
• 00:05:00 ultrasound
•500 µL transfer into clear vials
• Evaporate solvant in Genevac : 30 °C , 00:20:00 lipo mode
• Add 100 µL ISO:ACN 50:50 and transfer into insert Isopropanol HyperSolv CHROMANORMVWR International (Avantor)
QC pool preparation and dilutions
=>Pool 10 µL of each sample in amber vial (without insert) and prepare QC/2 QC/4
Boosting the QC pool with leucine 1ppm at 1/100
• Calibration range preparation: SST mix + Pest mix + PE (15:0) with fixed concentration IS metabo and Pest
25m
LIPOPHILIC FRACTION HPLC-MS/MS
• 00:05:00 ultrasound
• 125 µL transfer in insert in clear vials
• Dry evaporation under N2 flow at Room temperature
• Addition of the injection solvent for final dilution at 1/2: 250 µL of isopropanol + 50 µL IS polar lipids for subsequent analysis of glycolipids and phospholipids for example
5m
Protocol references
Giavalisco et al.2011 ; Sostare et al. 2018 ; Salem
et al. 2017 ; Cajka and Fiehn 2016 ; Pezzati et al.2019 ; Tufi
et al.2015