Jan 09, 2026
Version 1
Untargeted Lipidomics : Extraction of plasma / serum Lipids V.1
This protocol is a draft, published without a DOI.
- Emeline CHU-VAN1,
- Benoit Colsch1,
- florence castelli1
- 1CEA
- MetaboHub-IDF
External link: https://joliot.cea.fr/drf/joliot/Pages/Entites_de_recherche/medicaments_technologies_sante/SPI/lims.aspx
Protocol Citation: Emeline CHU-VAN, Benoit Colsch, florence castelli 2026. Untargeted Lipidomics : Extraction of plasma / serum Lipids. protocols.io https://protocols.io/view/untargeted-lipidomics-extraction-of-plasma-serum-l-hktpb4wmp
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 07, 2026
Last Modified: January 09, 2026
Protocol Integer ID: 238159
Keywords: serum lipids the extraction, lipids for untargeted lipidomic, serum lipidome, serum lipid, untargeted lipidomic, mtbe protein precipitation from plasma, internal standards in the extraction process, lipid, accurate comparison of relative lipid abundance, controlled extraction process, extraction of plasma, extraction process, mtbe protein precipitation, extraction, relative lipid abundance, serum
Funders Acknowledgements:
METABOHUB
Grant ID: 11-INBS-0010
Abstract
The extraction of lipids for untargeted lipidomics involves methanol/ MTBE protein precipitation from plasma / serum samples, aiming to maximize coverage of plasma / serum lipidome. The proposed protocol includes internal standards in the extraction process to answert to our quality control (QC) procedures. The expected outcome is a normalized and controlled extraction process, allowing for the accurate comparison of relative lipid abundances across all samples within the large cohorts.
Materials
Freezer -80°C :
Vortex shaker :
Centrifuge :
Ultrasonic processor :
plate reader (562 nm)
Evaporator :
Sharp scissors :
Lobind microtubes :
Micropipettes :
Protocol materials
tert-Butyl methyl ether, 99.9%, extra pureFisher ScientificCatalog #10474622
2-propanolMerckCatalog #34863
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
C12 Galactosyl(ß) Ceramide (d18:1/12:0)Avanti ResearchCatalog #860544
13:0 Lyso PEAvanti ResearchCatalog #856706
10:0 PSAvanti ResearchCatalog #840036
C12 Lactosyl(ß) Ceramide (d18:1/12:0)Avanti ResearchCatalog #860545
ChloroformMerckCatalog #132950
oleic acid-d9Avanti ResearchCatalog #861809
12:0 PGAvanti ResearchCatalog #840435
12:0 Dihydro SM (d18:0/12:0)Avanti ResearchCatalog #860683
17:0-17:1-17:0 D5 TGAvanti ResearchCatalog #860903
11:0 Lyso PCAvanti ResearchCatalog #855376
12:0 PEAvanti ResearchCatalog #850702
C12 Mono-Sulfo Galactosyl(ß) Ceramide (d18:1/12:0)Avanti ResearchCatalog #860573
13:0 Lyso PGAvanti ResearchCatalog #858126
13:0 PCAvanti ResearchCatalog #850340
Troubleshooting
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.).
Wear appropriate clothing, closed shoes, lab coat, glasses, gloves, extractor hood
Use of CMR solvents, use of nitrogen
Wear your Personal Protective Equipment (PPE) at least a lab coat, protective eyewear, gloves, long pants and closed toe shoes
Ethics statement
For experiments involving humans, the CPP (commission de protection des personnes physique) agreement.
Randomization
Reorganize stool samples in random order on dry ice (the same randomization is applied for the entire process (sample prep - LC-MS analysis - data treatment)
Store at -80 °C until extraction or proceed to aliquoting step.
Sample aliquoting and quality control sample constitution
Thaw plasma / serum samples.
Aliquoting : 50 µL or 25 µL depending on available volume in safe lock microtube
Create a quality control (QC) : pool of a same volume (~20 µL of all samples)
Aliquoting QC : 50 µL (or more) ependong on available volume
Store at -80 °C until sample extraction
Sample extraction
Thaw 50 µL plasma / serum aliquots.
The following steps are described for 50 µL samples.
Adjust the solvent extraction volumes according the aliquot volume.
Add 1 volume of MeOH containing Internal Standards 10x.
Extraction solvent :
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
IS mix :
| A | B | C | D | |
| For 4 mL of IS mix x10 (µL) | Final concentration (µg/mL) | |||
| std à 1mg/mL | ||||
| Optional | oleic acid-d9 | 200 | 50 | |
| L025 | 11:0 LPC | 100 | 25 | |
| L004 | 13:0 LPE | 40 | 10 | |
| L024 | 13:0 LPG | 400 | 100 | |
| L001 | 12:0 PG | 40 | 10 | |
| L016 | 13:0 PC | 500 | 125 | |
| L017 | 12:0 PE | 200 | 50 | |
| Optional | 10:0 PS | 400 | 100 | |
| L019 | d18:1/12:0 Cer | 220 | 55 | |
| L052 | d18:1/12:0 lacCer | 80 | 20 | |
| L021 | d18:1/12:0 GalCer | 240 | 60 | |
| L013 | d18:1/12:0 Su | 100 | 25 | |
| L022 | d18:0/12:0 SM | 60 | 15 | |
| L008 | 17:0-17:1-17:0 D5 TG | 200 | 50 |
all the solutions are prepared in Chloroform / Methanol 1/1 v/v (CM1/1)
oleic acid-d9Avanti ResearchCatalog #861809 (optional internal standard)C12 Galactosyl(ß) Ceramide (d18:1/12:0)Avanti ResearchCatalog #860544
11:0 Lyso PCAvanti ResearchCatalog #855376
13:0 Lyso PEAvanti ResearchCatalog #856706
13:0 Lyso PGAvanti ResearchCatalog #858126
12:0 PGAvanti ResearchCatalog #840435
13:0 PCAvanti ResearchCatalog #850340
12:0 PEAvanti ResearchCatalog #850702
10:0 PSAvanti ResearchCatalog #840036 (optional internal standard)
C12 Lactosyl(ß) Ceramide (d18:1/12:0)Avanti ResearchCatalog #860545
C12 Galactosyl(ß) Ceramide (d18:1/12:0)Avanti ResearchCatalog #860544
C12 Mono-Sulfo Galactosyl(ß) Ceramide (d18:1/12:0)Avanti ResearchCatalog #860573
12:0 Dihydro SM (d18:0/12:0)Avanti ResearchCatalog #860683
17:0-17:1-17:0 D5 TGAvanti ResearchCatalog #860903
IS mix solvent :
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
ChloroformMerckCatalog #132950
Vortex 1250 rpm 5 s
Add 3.6 volumes of MTBE
second extraction solvent :
tert-Butyl methyl ether, 99.9%, extra pureFisher ScientificCatalog #10474622
Vortex 1250 rpm 15 min
5000 x g, 5°C 15 min
Transfer supernatant in new safe lock microtube
Dry supernatants under a nitrogen stream at 40 °C using a
Equipment
TurboVap LV
NAME
Evaporator
TYPE
BIOTAGE
BRAND
TurboVap® LV
SKU
Add 200 µL CM1/1 for washing
Vortex 5 s
5000 rpm, 5°C 15 min
Transfer supernatants
Dry supernatants under a nitrogen stream at 40 °C using a
Equipment
TurboVap LV
NAME
Evaporator
TYPE
BIOTAGE
BRAND
TurboVap® LV
SKU
Reconstitute extracts with 50 µL CM1/1
Store at -20 °C until analysis
Dilution for LC-HRMS analysis
Dilute extracts (samples and QC) 100-fold in the mix :
65/35/5 Methanol / Isopropanol / H2O
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
2-propanolMerckCatalog #34863
Water LC-MS ChromasolvFisher ScientificCatalog #15665350
Transfer into glass vials (with glass insert)
Prepare 2-fold, 4-fold and 8-fold diluted QC samples
LC-MS analysis
LC conditions and parameters :
Mobile phases
Mobile phase A :
60/40 H2O/MeOH + 0.1% HCOOH + 10 mM ammo formate
1200 mL ultrapure water
800 mL Methanol
2 mL formic acid 100%
1.2612 g ammonium formate
mix
Mobile phase B :
90/10 IPA/MeOH + 0.1% HCOOH + 10 mM ammo formate
200 mL Methanol
1.2612 g ammonium formate
ultrasonic bath during 5 min
1800 mL 2-propanol
mix
Chromatographic column
Flow rate = 0.4 mL/min
Kinetex C8 - 2.6 µm - 100 Å - 150 x 2.1 mm
Equipment
Kinetex C8
NAME
LC column
TYPE
Phenomenex
BRAND
00F-4497-AN
SKU
LINK
The column is protected with an on-line filter.
On-line filter parts :
Equipment
Replacement Tips
NAME
HPLC guard column accessoire
TYPE
ThermoFisher Scientific
BRAND
850-RT
SKU
LINK
Equipment
Replacement Filters
NAME
LC accessoire
TYPE
Thermo Scientific
BRAND
22017
SKU
LINK
Equipment
Direct-Connection Filter System Components
NAME
LC accessoire
TYPE
Thermo Fisher Scientific
BRAND
27002
SKU
LINK
Elution gradient
| Time (min) | mobile phase A (%) | mobile phase B (%) | |
| 0.0 | 68 | 32 | |
| 2.5 | 55 | 45 | |
| 3.5 | 48 | 52 | |
| 5.0 | 42 | 58 | |
| 7.0 | 34 | 66 | |
| 10.0 | 30 | 70 | |
| 13.0 | 25 | 75 | |
| 16.0 | 20 | 80 | |
| 20.0 | 15 | 85 | |
| 23.0 | 10 | 90 | |
| 26.0 | 5 | 95 | |
| 28.0 | 68 | 32 | |
| 42.0 | 68 | 32 |
Temperatures
Oven temperature 60 °C
Autosampler temperature 5 °C
MS conditions and parameters
For high resolution mass spectrometer (Exploris/Orbitrap like)
Ion source type : HESI
Spray voltage : 3700 V (positive mode) / 3200 V (negative mode)
Sheath gas : 50
Auxiliary gas : 15
Sweep gas : 1
Ion transfer tube temperature : 360°C
Vaporizer temperature : 300°C
Orbitrap resolution : 240 000
Scan range (m/z) : 150 - 2000
RF lens (%) : 70