Jan 31, 2025

Public workspaceBiphasic extraction metabolites in algal matrix or periphyton

Forked from a private protocol
This protocol is a draft, published without a DOI.
  • 1INRAE
  • MetaboHUB-Bordeaux
  • MetaboHUB
Mélissa ME EON
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External link: https://metabolome.u-bordeaux.fr/en/plateforme-bordeaux-metabolome-english/
Protocol Citation: Mélissa ME EON, NicolasCreusot 2025. Biphasic extraction metabolites in algal matrix or periphyton. protocols.io https://protocols.io/view/biphasic-extraction-metabolites-in-algal-matrix-or-dgwn3xde
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: July 09, 2024
Last Modified: January 31, 2025
Protocol Integer ID: 103086
Keywords: extraction, metabolites, environnemental metabolomics
Funders Acknowledgements:
MetaboHUB
Grant ID: ANR-11-INBS-0010
Bordeaux Metabolome Platform
Grant ID: https://doi.org/10.15454/1.5572412770331912E12
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 Temperature-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.


Biphasic extraction metabolites in algal and biofilm matrix

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
https://www.fishersci.com/shop/products/2-5l-84c-bt-fd-sst-115v-60hz/10400014
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
https://uk.mpbio.com/fastprep-24-5g-instrument
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
ReagentMTBE: Sigma Chromasolv 99.8% for HPLC 100mL (smallest available) (34875-100mL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34875-100ML
ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641
ReagentMilliQ water
ReagentAcetonitrile
ReagentIsopropanol (IPA) 100%
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 recycled according to the quality document in force at the EABX_GEN_706 laboratory.

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

Extraction steps overview

PREPARATION
PREPARATION
2h
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) =>Amount150 mL stored at Temperature4 °C for one week
• Prepare a mixture H2O:MeOH (3:1, v:v) => Amount100 mL stored at Temperature4 °C for one week
ReagentMTBE: Sigma Chromasolv 99.8% for HPLC 100mL (smallest available) (34875-100mL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34875-100ML ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641
ReagentMilliQ waterContributed by users
PREPARATION OF BALL TUBES AND VIALS
All these steps can be done the day before.
• Annotate all tubes
• Add Amount150 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 Temperature-80 °C as well as microtube racks
PREPARATION OF MIXTURES FOR DOPING:
SST mix (Concentration50 Parts per Million (PPM) ) + pesticide mix (Concentration3.3 Parts per Million (PPM) )mix IS pesticides (Concentration2.5 Parts per Million (PPM) ) + mix IS metabo (Concentration50 Parts per Million (PPM) ) + PE (15:0) (Concentration100 Parts per Million (PPM) )
• Ultrasound Duration00:05:00 at TemperatureRoom temperature

5m
EXTRACTION STEPS
EXTRACTION STEPS
2h
2h
PRELIMINARY STEPS
On ice, in the soundcheck room:
• Add Amount10 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: Amount10 µL IS pesticides + Amount10 µL IS metabo + Amount50 µL PE (15:0)
o Lab sample: Amount10 µL IS of the test compound + Amount10 µL IS metabo + Amount10 µL PE (15:0)
o Spike on matrix (or solvent if matrix not available): Extraction yield
Spike 1:
• Pre-extraction: Amount10 µL mix SST + Amount10 µL mix Pest + PE (15:0)
• Post-extraction: Amount10 µL IS metabo + Amount10 µL IS pesticides
Spike 2:
• Post-extraction: Amount10 µL IS metabo +Amount10 µL IS pesticides
o Procedural blank: solvent only
o Blind blank: solvent + Amount10 µL All IS

In order to guarantee the cold chain:
• For Duration00:15:00 : Place the tubes and MTBE:MeOH solution at Temperature-80 °C ; H20:MeOH solutions at Temperature-20 °C
• Start the Fast Temp on the centrifuge Temperature4 °C



15m
EXTRACTION 1
Under hood and on ice: Add Amount1 mL of MTBE:MeOH (3:1) per tube
• Cycle to Fast prep:Duration00:00:15 at 6.5 nm
• Place the samples in ice or at Temperature-80 °C for Duration00:03:00
• Do a second cycle in Fast prep: Duration00:00:15 at 6.5 nm
• Place the samples in ice or at Temperature-80 °C for Duration00:03:00

Under hood and on ice: Add Amount650 µL of H2O:MeOH
• Do a third cycle in Fast prep: Duration00:00:15 at 6.5 nm
• Place the samples in ice or at Temperature-80 °C for Duration00:03:00
• Centrifugation: Centrifigation12000 rpm, 4°C, 00:05:00

Under the hood and on ice, recover the phases in vials stocks (amber 1.8mL)
=>Amount500 µL lipophilic above; Amount600 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
https://uk.mpbio.com/fastprep-24-5g-instrument
LINK

14m 45s
EXTRACTION 2
Under hood and on ice: Add Amount700 µL of MTBE:MeOH (3:1) per tube
• Cycle to Fast prep:Duration00:00:15 at 6.5 nm
• Place the samples in ice or at Temperature-80 °C for Duration00:03:00
• Do a second cycle in Fast prep: Duration00:00:15 at 6.5 nm
• Place the samples in ice or at Temperature-80 °C for Duration00:03:00

Under hood and on ice: Add Amount455 µL of H2O:MeOH
Do a third cycle in Fast prep: Duration00:00:15 at 6.5 nm
• Place the samples in ice or at Temperature-80 °C for Duration00:03:00
Centrifugation: Centrifigation12000 rpm, 4°C, 00:05:00
Under the hood and on ice, recover the phases under the hood in the same vials
=>Amount600 µL lipophilic above;Amount700 µL hydrophilic below using a micropipette

End of the process
• Weigh the amber stocks after transfer of the extracts
=>Theoretically: Amount1.1 mL lipo vs Amount1.3 mL hydro
•Doping
o Spike 1: Extraction yield
• Post-extraction:Amount10 µL IS metabo + Amount10 µL IS pesticides
o Spike 2:
• Post-extraction: Amount10 µL IS metabo +Amount10 µL IS pesticides
• Store all vials atTemperature-80 °C

14m 45s
PREPARATION OF VIALS FOR INJECTION
PREPARATION OF VIALS FOR INJECTION
2h
2h
HYDROPHILIC FRACTION UPLC-TOF
• Duration00:05:00 ultrasound
•Amount500 µL transfer into clear vials
• Evaporate solvant in Genevac : Temperature30 °C , Duration00:30:00 hydro mode
• Add Amount100 µL H2O:ACN 50:50 and transfer into insert
ReagentAcetonitrile Contributed by users
• QC pool preparation and dilutions
=>Pool Amount10 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
• Duration00:05:00 ultrasound
•Amount500 µL transfer into clear vials
• Evaporate solvant in Genevac : Temperature30 °C , Duration00:20:00 lipo mode
• Add Amount100 µL ISO:ACN 50:50 and transfer into insert
ReagentIsopropanol (IPA) 100%Contributed by users
QC pool preparation and dilutions
=>Pool Amount10 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
• Duration00:05:00 ultrasound
• Amount125 µL transfer in insert in clear vials
• Dry evaporation under N2 flow at TemperatureRoom temperature
• Addition of the injection solvent for final dilution at 1/2: Amount250 µL of isopropanol + Amount50 µL IS polar lipids for subsequent analysis of glycolipids and phospholipids or 500 μL of a mixture of "PMA and PMB" + IS neutral lipids for DGTS and triglycerides.

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