Oct 30, 2025
Version 2
Expanding the clinical use of DBS: through a metabolomic and a lipidomic evaluation_Mat&Meth V.2
- Jérémy nteiro1,2,3,
- cyrielle.clement 4,
- Antoine Lefèvre1,2
- 1Université de Tours, INSERM, Imaging Brain & Neuropsychiatry iBraiN U1253, 37032, Tours, France;
- 2Plateforme de Métabolomique et d'Analyses Chimiques, US61 ASB, Université de Tours, CHRU Tours, Inserm, Tours, France;
- 3MetaboHUB-Tours, Tours, France;
- 4MetaboHUB-MetaToul-Lipidomique, MetaboHUB-ANR-11-INBS-0010, Inserm U1297, Université Paul Sabatier Toulouse III, 31432 Toulouse, France
Protocol Citation: Jérémy nteiro, cyrielle.clement , Antoine Lefèvre 2025. Expanding the clinical use of DBS: through a metabolomic and a lipidomic evaluation_Mat&Meth. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbwkyqgpk/v2Version created by Jérémy Monteiro
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: October 28, 2025
Last Modified: October 30, 2025
Protocol Integer ID: 230969
Keywords: metabolomics, lipidomics, targeted, untargeted, quantification, extraction, serum, Dried Blood Spot, biological information, LC-MS, SFC-MS, lipidome of serum, screening of metabolome, clinical use of db, metabolomic, metabolome, metabolite, lipidome, lipid, serum, clinical use, db, extraction to the data analysis
Funders Acknowledgements:
Agence Nationale de la Recherche
Grant ID: ANR-11-INBS-0010 MetaboHUB
Agence Nationale de la recherche au titre de France 2030
Grant ID: ANR-21-ESRE-0035
Disclaimer
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Abstract
WARNING : this protocols is connect to the corresponding research paper about DBS and serum.
The only authors described here are the one which have developped or carry out the analysis.
In this protocole, multiple analysis are described to quantify metabolites and lipids & screening of metabolome and lipidome of serum and DBS. For every analysis, the whole process is described, from the extraction to the data analysis.
If some information are missing, please report to the corresponding research paper, in the Mat&Meth section or in the supplementary data.
Image Attribution
Laurent Galineau (Plateforme Imagerie Préclinique, US-61 ASB, Université de Tours, CHRU Tours, Inserm, Tours, France ; Université de Tours, INSERM, Imaging Brain & Neuropsychiatry iBraiN U1253, 37032, Tours, France)
Camille Dupuy (Plateforme de Métabolomique et d'Analyses Chimiques, US61 ASB, Université de Tours, CHRU Tours, Inserm, Tours, France; MetaboHUB-Tours, Tours, France; Université de Tours, INSERM, Imaging Brain & Neuropsychiatry iBraiN U1253, 37032, Tours, France)
Guidelines
Metabolomics is the “omics” science studying metabolites and can meet the need for a deeper insight into biological data. It offers a real-time snapshot of a metabolic state and so reflect immediate physiological changes.
By revealing metabolic profiles, metabolomics allows for precise diagnosis, tailored treatments, and proactive health management, ensuring highly personalized and effective interventions.
(Nordström, A., Lewensohn, R. Metabolomics: Moving to the Clinic. J Neuroimmune Pharmacol 5, 4–17 (2010). https://doi.org/10.1007/s11481-009-9156-4 )
Materials
Mass Spectrometers
- Q-Exactive or equivalent
- Xevo TQ-XS or Xevo TQ-A
- SFC-G2-TOF or equivalent
Others
- agitators
- micro-pipettes (1000 µL, 200 µL, 20 µL)
- planar agitator
- centrifuge
- SpeedVac
- N2-flow evaporator
- falcon
- hemolyse tube
- eppendorf (1.5 mL)
- glass vial (2 mL)
Protocol materials
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
Isopropanol HyperSolv CHROMANORMVWR International (Avantor)
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor)
Methanol HiPerSolv CHROMANORMVWR International (Avantor)
formiate ammoniumVWR International (Avantor)
Aluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich)
Calibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher
Calibration Solution Pierce ESI Negative IonThermo Fisher
IROA MSMLS, OAMLS, BACSMLS, FAMLSMerck MilliporeSigma (Sigma-Aldrich)
L-tryptophanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T0254
Indoxyl-3-sulfateMerck MilliporeSigma (Sigma-Aldrich)
Kynurenic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #K3375
indole-3-Lactic AcidMerck MilliporeSigma (Sigma-Aldrich)
water LC-MS ChromasolvFisher ScientificCatalog #15665350
Ammonium acetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G
3-nitrophenylhydrazineMerck MilliporeSigma (Sigma-Aldrich)
acetic-13C2-acidToronto Research Chemicals Inc
butanoic-d7-acidToronto Research Chemicals Inc
valeric-d9-acidToronto Research Chemicals Inc
n-(3-dimethylaminopropyl)-n-ethylcarboiimideMerck MilliporeSigma (Sigma-Aldrich)
3-indolepropionic-d2-acidToronto Research Chemicals Inc
melatonin d4Toronto Research Chemicals Inc
3-hydroxykynurenine 13C2,15N1Toronto Research Chemicals Inc
L-kynurenine d4Toronto Research Chemicals Inc
5-hydroxy-L-tryptophan d4Toronto Research Chemicals Inc
1H-indole-d5-3-acetamideToronto Research Chemicals Inc
indole-3-aldehyde 13C8Toronto Research Chemicals Inc
3-hydroxyanthranilic-d3-acidToronto Research Chemicals Inc
tryptamine d4Toronto Research Chemicals Inc
L-tryptophan d5Toronto Research Chemicals Inc
serotonin d4Toronto Research Chemicals Inc
indoxyl-13C6 sulfate potassium saltToronto Research Chemicals Inc
5-hydroxyindole-3acetic-d5-acidToronto Research Chemicals Inc
indoleacetic-d4-acidToronto Research Chemicals Inc
tryptophaneMerck MilliporeSigma (Sigma-Aldrich)
tryptophol d4Toronto Research Chemicals Inc
picolinic-d3-acidToronto Research Chemicals Inc
kynurenic-d5-acidToronto Research Chemicals Inc
Quinolinic-d3-acidToronto Research Chemicals Inc
N-acetylserotonine d3Toronto Research Chemicals Inc
indole-3-aldehydeMerck MilliporeSigma (Sigma-Aldrich)
melatonineMerck MilliporeSigma (Sigma-Aldrich)
5-hydroxyindole acetic acidToronto Research Chemicals Inc
picolinic acidMerck MilliporeSigma (Sigma-Aldrich)
indoxyl-3-sulfateToronto Research Chemicals Inc
indole-3-lactic acidMerck MilliporeSigma (Sigma-Aldrich)
5-hydroxytryptophaneMerck MilliporeSigma (Sigma-Aldrich)
kynurenineMerck MilliporeSigma (Sigma-Aldrich)
3-hydroxyanthranilic acidMerck MilliporeSigma (Sigma-Aldrich)
N-acetylserotonineMerck MilliporeSigma (Sigma-Aldrich)
quinolinic acidMerck MilliporeSigma (Sigma-Aldrich)
3-hydroxykynurenineMerck MilliporeSigma (Sigma-Aldrich)
xanthurenic acidMerck MilliporeSigma (Sigma-Aldrich)
tryptopholMerck MilliporeSigma (Sigma-Aldrich)
indole-3-acetic acidMerck MilliporeSigma (Sigma-Aldrich)
indole-3-propionic acidMerck MilliporeSigma (Sigma-Aldrich)
serotoninMerck MilliporeSigma (Sigma-Aldrich)
tryptamineMerck MilliporeSigma (Sigma-Aldrich)
indole-3-acetamideMerck MilliporeSigma (Sigma-Aldrich)
kynurenic acidMerck MilliporeSigma (Sigma-Aldrich)
deoxycholic acid d5Toronto Research Chemicals Inc
lithocholic acid d4Toronto Research Chemicals Inc
cholic acid d5Toronto Research Chemicals Inc
cholic acid sulfate d4Toronto Research Chemicals Inc
glycocholic acid d4Toronto Research Chemicals Inc
taurocholic acid d4Toronto Research Chemicals Inc
ursodeoxycholic acid d4Toronto Research Chemicals Inc
chenodeoxycholic acid d4Toronto Research Chemicals Inc
Water milliQMerck MilliporeSigma (Sigma-Aldrich)
acetic acidMerck MilliporeSigma (Sigma-Aldrich)
propionic acidMerck MilliporeSigma (Sigma-Aldrich)
butyric acidMerck MilliporeSigma (Sigma-Aldrich)
isobutyric acidMerck MilliporeSigma (Sigma-Aldrich)
valeric acidMerck MilliporeSigma (Sigma-Aldrich)
isovaleric acidMerck MilliporeSigma (Sigma-Aldrich)
taurochenodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
taurohyocholic acidMerck MilliporeSigma (Sigma-Aldrich)
glycohyocholic acidMerck MilliporeSigma (Sigma-Aldrich)
taurodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
glycodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
lithocholic acidMerck MilliporeSigma (Sigma-Aldrich)
glycochenodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
lithocholic acid sulfateMerck MilliporeSigma (Sigma-Aldrich)
taurolithocholic acidMerck MilliporeSigma (Sigma-Aldrich)
cholic acidMerck MilliporeSigma (Sigma-Aldrich)
glycolithcholic acidMerck MilliporeSigma (Sigma-Aldrich)
cholic acid sulfateMerck MilliporeSigma (Sigma-Aldrich)
taurocholic acidMerck MilliporeSigma (Sigma-Aldrich)
deoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
ursodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
hyocholic acidMerck MilliporeSigma (Sigma-Aldrich)
glycoursodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
tauroursodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
chenodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
glycocholic acidMerck MilliporeSigma (Sigma-Aldrich)
aconitic acidMerck MilliporeSigma (Sigma-Aldrich)
fumaric acidMerck MilliporeSigma (Sigma-Aldrich)
malic acidMerck MilliporeSigma (Sigma-Aldrich)
citric acidMerck MilliporeSigma (Sigma-Aldrich)
isocitric acidMerck MilliporeSigma (Sigma-Aldrich)
ketoglutaric acidMerck MilliporeSigma (Sigma-Aldrich)
succinic acidMerck MilliporeSigma (Sigma-Aldrich)
itaconic acidMerck MilliporeSigma (Sigma-Aldrich)
lactic acidMerck MilliporeSigma (Sigma-Aldrich)
Troubleshooting
Problem
Retreving a punch from a dry blank blotter paper
Solution
Soaked them with LC-MS grade water and then punch them
Problem
Normalisation of quantification in DBS
Solution
Not used in this study but if you can, measured the hematocrit level of every patient during sampling session
Problem
DBS storage
Solution
Dedicate a space at -80°C or -20°C for your DBS with a desiccator
Problem
Contamination with 3-nitrophenylhydrazine
Solution
LC-MS system: be careful if you shrink gradient length
Pipettes: avoid (if possible) to use only 1 pipette for all --> prefer one pipette per type of solution
Safety warnings
Be really carefull with DBS handling (no contact with skin, don't breath on it) and storage (humidity, UV exposition, temperature and long-time storage).
Ethics statement
DBS and serum samples were collected at the same time, from 4 healthy patients. All samples were obtained in accordance with French legislation (Loi Jardé). In French hospitals with a research mission, the use of sample leftovers for research purposes is based on the principle of no opposition. According to Loi Jardé, no requirement of Ethical Committee or Institutional Review Board was required because no new sampling was required. This law also stipulates that patients must be fully informed of this information/practice and patients must have the possibility, at any time, of objecting to the use of their samples in research. The analyses were always performed in accordance with confidentiality rules. Data were coded without mention of first and last names, and the results were produced in a way that does not allow patients to be identified. Patients did not invoke their right to have a right of access, rectification, portability and limitation of the processing of data and/or biological samples.
Before start
- Dried Blood Spot (DBS) represent the whole blank blotter paper with several blood soaked spots : only these spots aree interesting so it is mendatory to retrieve them as 3 mm diameter disc called a punch, with specific equipment : a puncher (available in mostly all hospitals)
- If you are going to do some comparison between DBS and other blood sample, try to have all you sample from same patient(s)
A.1. Large Targeted Metabolomics
8h
Extraction of DBS
In the whole DBS card Whatman 903TM, retrieved 2 punches (1 punch = 3 mm disc) with a puncher
WARNINGS:
- a punch must be entirely soaked in blood
- if possible, do not punch near the exterior of the blood droplets of the DBS
Put the 2 punches in a 1.5 mL Eppendorf vial
Add 200 µL of Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (4:4:2)
Vigourously vortexed during 00:01:00
1m
Planar agitation : 01:00:00 ,Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
1h
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
10m
Transfert 150 µL of supernateant in a glass vial of 1.5 mL
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Dry for 01:00:00 under vacuum at 40 °C
1h
Dried extract is redissolved with 75 µL of:
- For reversed-phase analysis: Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (2:8)
- For HILIC analysis: Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (8:2)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigorously vortexed during 00:01:00 at Room temperature
Planar agitation: 00:30:00 , Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
Transfert whole aliquot in a 200 µL 96-well plates (1 for reversed-phase; 1 for HILIC)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Injection Volume (µL) : 10
Extraction of Serum
Make aliquots of 50 µL of serum
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Add 150 µL of cold Methanol HiPerSolv CHROMANORMVWR International (Avantor) (-20 °C )
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
Let it 00:20:00 at -20 °C
20m
Planar agitation : 01:00:00 ,Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 150 µL of supernateant in a glass vial of 1.5 mL
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Dry for 01:00:00 under vacuum at 40 °C
Dried extract is redissolved with 100 µL of:
- For reversed-phase analysis: Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (2:8)
- For HILIC analysis: Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (8:2)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigorously vortexed during 00:01:00 at Room temperature
Planar agitation: 00:30:00 , Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
Transfert whole aliquot in a 200 µL 96-well plates (1 for reversed-phase; 1 for HILIC)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Injection Volume (µL) : 5
LC parameters #1 - Reversed-Phase
Phase A:
- 200 mL + 5.5 mL * [Number of injections]
- Water milliQMerck MilliporeSigma (Sigma-Aldrich) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Phase B:
- 200 mL + 3.8 mL * [Number of injections]
- Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Chromatographic System
Equipment
Ultimate 3000
NAME
UHPLC
TYPE
Dionex Softron
BRAND
SRD-3400
SKU
Pump, Column Compartment, AutoSampler
SPECIFICATIONS
Install the column in the oven
Equipment
SecurityGuard ULTRA Cartridges
NAME
UPLC C18 2.1mm ID Columns
TYPE
Phenomenex
BRAND
AJ0-8782
SKU
Equipment
Kinetex 1.7µm XB-C18 100A
NAME
Column
TYPE
Phenomenex
BRAND
00F-4498-AN
SKU
LC Column 150 x 2.1mm
SPECIFICATIONS
Adjust the temperature (55 °C ) and let it heat the core of the column
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Purge the system:
- 1 time at 100%A and 1 time at 100%B with open valve
- 2 times with open valve, 2 times with close valve at 50%A and 50%B
- Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
- Check that the pression is stable; otherwise : purge again
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 2%B
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0 | 0.5 | 2 | |
| 0 | 0.5 | 2 | |
| 2 | 0.5 | 2 | |
| 6 | 0.5 | 30 | |
| 9 | 0.5 | 75 | |
| 9.5 | 0.5 | 100 | |
| 11.8 | 0.5 | 100 | |
| 12 | 0.5 | 2 | |
| 13.7 | 0.5 | 2 |
LC parameters #2 - HILIC
Phase A:
- 200 mL + 2.6 mL * [Number of injections]
- Water milliQMerck MilliporeSigma (Sigma-Aldrich) + 10 millimolar (mM) formiate ammoniumVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Phase B:
- 200 mL + 3.8 mL * [Number of injections]
- [95% of
- Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) & 5% of Water milliQMerck MilliporeSigma (Sigma-Aldrich) ] + 10 millimolar (mM) formiate ammoniumVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Chromatographic System
Equipment
Ultimate 3000
NAME
UHPLC
TYPE
Dionex Softron
BRAND
SRD-3400
SKU
Pump, Column Compartment, AutoSampler
SPECIFICATIONS
Install the column in the oven
Equipment
CORTECS UPLC HILIC 1.6µM
NAME
Column
TYPE
Waters
BRAND
Adjust the temperature (40 °C ) and let it heat the core of the column
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Purge the system:
- 1 time at 100%A and 1 time at 100%B with open valve
- 2 times with open valve, 2 times with close valve at 50%A and 50%B
- Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
- Check that the pression is stable; otherwise : purge again
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 95%B
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0 | 0.5 | 95 | |
| 0 | 0.5 | 95 | |
| .9 | 0.5 | 95 | |
| 4.8 | 0.5 | 82 | |
| 9 | 0.5 | 10 | |
| 11.2 | 0.5 | 10 | |
| 11.4 | 0.5 | 95 | |
| 13.7 | 0.5 | 95 |
Mass Spectrometry
Mass Spectrometer
Equipment
Q-Exactive
NAME
Mass Spectrometer
TYPE
ThermoScientific
BRAND
HESI Source
SPECIFICATIONS
MS Parameters :
- Scan Range: 58-870 m/z
- Resolution MS1: 70,000
- Resolution MS2: 15,000
- Polarity: POS or NEG
- Microscans: 1
- Lock mass: None
- AGC Target: 1E6
- Maximum Inject Time: 250
- Sheath Gas Flow Rate: 50
- Aux Gas Flow rate: 15
- Sweep Gas flow Rate: 1
- Spray Voltage (kV): +/-2.5
- Capillary Temperature (°C): 325
- S-lens RF level: 60
- Aux gas Heater temperature (°C): 400
Cleaning the source:
- Set source temperature to 50 °C
- Let it "cool" 01:00:00
- Unplug the Ion MAx API source with HESI probe attached
- Unscrewed the Sweep Cone and the capillary
- Immediately plug another cleaned capillary
- Clean the sweep cone with wet Aluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich)
- Rinse thoroughly with Water milliQMerck MilliporeSigma (Sigma-Aldrich)
- In a glass beaker, cover completely the Sweep Cone and the capillary with Methanol HiPerSolv CHROMANORMVWR International (Avantor)
- Ultrasound during 00:10:00
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
- Let it dry
- Replace the cone and the Ion Max API source with HESI probe
- Set source temperature back to normal
Reinstall the source & allow it to return to analysis conditions
Calibration:
- POS: Calibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher
- NEG: Calibration Solution Pierce ESI Negative IonThermo Fisher
- RMS < 0.4/0.4 in both ionisation mode
Equipment
Fusion 100T
NAME
syringe pump
TYPE
Chemyx Inc.
BRAND
Prepared a solution for Performance Evaluation (during sequence injection):
- Solvent : Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:1)
- L-tryptophanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T0254 at 10000 nanomolar (nM)
- Indoxyl-3-sulfateMerck MilliporeSigma (Sigma-Aldrich) at 3750 nanomolar (nM)
- Kynurenic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #K3375 at 25 nanomolar (nM)
- indole-3-Lactic AcidMerck MilliporeSigma (Sigma-Aldrich) at 500 nanomolar (nM)
Once calibrated, sequences can be run
Typical Sequences:
| Sample Type | Injection Volume (µL) | |
| Equilibrium(=sample) * 10 | 5 | |
| Blank Solvent | 5 | |
| Performance Evaluation * 3 | 5 | |
| Blank Solvent | 5 | |
| Sample QC | 5 | |
| Sample *10 | 5 | |
| Sample QC | 5 | |
| ... | 5 | |
| Last Sample QC | 5 | |
| Blank Solvent * 3 | 5 |
Data Analysis
Metabolite Identification :
- m/z and RT of a chemical library (IROA MSMLS, OAMLS, BACSMLS, FAMLSMerck MilliporeSigma (Sigma-Aldrich) ) checked with
Software
Thermo Scientific Xcalibur
NAME
- MS² acquired on QC samples
- File exported : list of m/z and RT
Features detection:
- Peak Picking on all samples is done by using Workflow4Metabolomics
- Automatic attribution of the precedent list
Software
Workflow4Metabolomics
NAME
doi: 10.1093/bioinformatics/btu813
DEVELOPER
REPOSITORY
SOURCE LINK
Fusion of modalites = sorting redundancies:
- Only the best modality is kept for 1 metabolite, based on RT > Dead Volume (0-1 min) and/or lower CV(metabolite) on QCs
A.2. Quantification of Short-Chain Fatty Acids (SCFA)
6h
Extraction of DBS
In the whole DBS card Whatman 903TM, retrieved 2 punches (1 punch = 3 mm disc) with a puncher
WARNINGS:
- a punch must be entirely soaked in blood
- if possible, do not punch near the exterior of the blood droplets of the DBS
Put the 2 punches in a 1.5 mL Eppendorf vial
Add 200 µL of Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (4:4:2)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
Planar agitation : 01:00:00 ,Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
50 µL of supernateant is derived in a 1 mL 96-wells plate
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Extraction of Serum
Make aliquots of 50 µL of serum
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Add 150 µL of cold Methanol HiPerSolv CHROMANORMVWR International (Avantor) (-20 °C )
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
Let it 00:20:00 at -20 °C
Planar agitation : 01:00:00 ,Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
50 µL of supernateant is derived in a 1 mL 96-wells plate
Calibration Curve
Prepared independant 1mg/mL solution for acetic acidMerck MilliporeSigma (Sigma-Aldrich) , propionic acidMerck MilliporeSigma (Sigma-Aldrich) , butyric acidMerck MilliporeSigma (Sigma-Aldrich) ,isobutyric acidMerck MilliporeSigma (Sigma-Aldrich) ,valeric acidMerck MilliporeSigma (Sigma-Aldrich) and isovaleric acidMerck MilliporeSigma (Sigma-Aldrich)
These solutions are called SM.
Then, prepared "SG0" solution :
| A | B | C | |
| Metabolite | Volume of SM required (µL) | Concentration SGO (pmol/mL) | |
| Acetic acid | 72 | 300000.00 | |
| Butyric acid | 3.5 | 10000.00 | |
| Isobutyric acid | 3.5 | 10000.00 | |
| Isovaleric acid | 4.1 | 10000.00 | |
| Propanoic acid | 5.9 | 20000.00 | |
| Valeric acid | 4.1 | 10000.00 | |
| Vtot SM (µL) | 93 | / | |
| V(H2O) (µL) | 3 907 | / |
Prepared SG1 solution, which is a dilution by 100 of SG0
Calibration Curve:
| A | B | C | D | E | F | G | H | |
| From which ? | SG1 | SG1 | SG1 | SG0 | SG0 | SG0 | SG0 | |
| Calibration Curve Solutions | L1 | L2 | L3 | L4 | L5 | L6 | L7 | |
| V(SG), µL | 75 | 250 | 500 | 25 | 75 | 250 | 500 | |
| V(H2O), µL | 925 | 750 | 500 | 975 | 925 | 750 | 500 |
Preparation of calibration curve solutions (a solution si named Lx; x from 1 (less concentrated) to 7 (most concentrated))
| A | B | C | D | |
| Solution | QC Low | QC Middle | QC High | |
| V(SG), µL | 20 | 200 | 400 | |
| V(H2O), µL | 980 | 800 | 600 |
Preparation of calibration curve QC, all from SG0
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Derivatization
All information are available in the following papers:
Citation
LINK
Before derivatization reactions, the following solutions must be prepared in a volume > [50 µL * number of sample]:
- (A) An internal standard solution of acetic-13C2-acidToronto Research Chemicals Inc ,butanoic-d7-acidToronto Research Chemicals Inc and valeric-d9-acidToronto Research Chemicals Inc at 1 nM in Water milliQMerck MilliporeSigma (Sigma-Aldrich)
- (B) A 2.5% pyridine solution in Methanol HiPerSolv CHROMANORMVWR International (Avantor)
- (C) A 105 nM solution of EDC (n-(3-dimethylaminopropyl)-n-ethylcarboiimideMerck MilliporeSigma (Sigma-Aldrich) ) in Methanol HiPerSolv CHROMANORMVWR International (Avantor)
- (D) A 175 nM 3-NPH (3-nitrophenylhydrazineMerck MilliporeSigma (Sigma-Aldrich) ) solution in Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:1)
Derivatization reaction:
- Successively add to your sample 50 µL of A, B C and D, in this order
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Let react 01:00:00 at 4 °C
Quench the reaction with 250 µL of acidified water (1 % of Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260 in Water milliQMerck MilliporeSigma (Sigma-Aldrich) )
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Cover the vials or the 96-wells plate
LC parameters
Phase A:
- Water milliQMerck MilliporeSigma (Sigma-Aldrich) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Phase B:
- Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Chromatographic System
Equipment
Acquity I-Class UPLC
NAME
Chromatography
TYPE
Waters
BRAND
Install the column in the oven
Equipment
Kinetex 1.7µm XB-C18 100A
NAME
Column
TYPE
Phenomenex
BRAND
00F-4498-AN
SKU
LC Column 150 x 2.1mm
SPECIFICATIONS
Adjust the temperature (55 °C ) and let it heat the core of the column
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Purge the system:
- 1 time at 100%A and 1 time at 100%B with open valve
- 2 times with open valve, 2 times with close valve at 50%A and 50%B
- Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
- Check that the pression is stable; otherwise : purge again
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 2%B
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0 | 0.6 | 15 | |
| 0 | 0.6 | 15 | |
| 2 | 0.6 | 15 | |
| 4 | 0.6 | 25 | |
| 6 | 0.6 | 40 | |
| 8 | 0.6 | 50 | |
| 9 | 0.6 | 85 | |
| 9.5 | 0.6 | 0 | |
| 12.4 | 0.6 | 0 | |
| 14.5 | 0.6 | 15 |
Injection volume : 5 µL for both samples
Mass Spectrometry
Mass Spectrometer
Equipment
Xevo TQ-XS, with an ESI probe
NAME
Mass Spectrometer
TYPE
Waters
BRAND
MS Parameters :
- MRM mode
- Polarity : NEG
- Capillary Voltage : 3 kV
- Desolvatation Temperature: 450 °C
- Source Temperature: 150 °C
- Nebuliseur Gas Flow: 7 bar
- COne Gas Flow Temperature: 150°C
- Desolvatation Gas Flow: 1200 L/hr
MS method
| Metabolite | RT(min) | Parent Ion (m/z) | Daugther Ion (m/z) | CE(-eV) | ConeVoltage (V) | Internal Standard | Dynamic Range ofConcentration (nM) | LOQa²(nM) | CO (%) | |
| Acetic Acid | 1.67 | 194.1 | 152.01 | 14 | 20 | Acetic-13C2-Acid | 638 - 425,000 | 0.42 | 4.47 | |
| Propionic Acid | 2.50 | 208.1 | 165.1 | 16 | 20 | Butyric-d7-Acid | 300 - 200,000 | 0.27 | 6.53 | |
| Isobutyric Acid | 3.60 | 222.1 | 179.1 | 14 | 20 | Butyric-d7-Acid | 30 - 20,000 | 0.04 | 13.22 | |
| Butyric Acid | 3.73 | 222.1 | 179.1 | 14 | 20 | Butyric-d7-Acid | 338 - 225,000 | 0.42 | 0.62 | |
| Isovaleric Acid | 5.25 | 236.1 | 193.1 | 14 | 20 | Valeric-d9-Acid | 18 - 12,000 | 0.12 | 15.76 | |
| Valeric Acid | 5.50 | 236.1 | 193.1 | 14 | 20 | Valeric-d9-Acid | 140 - 46,500 | 0.10 | 2.67 | |
| Acetic-13C2-Acid | 1.67 | 196.1 | 152.01 | 14 | 20 | - | - | - | - | |
| Butyric-d7-Acid | 3.72 | 229.1 | 186.1 | 14 | 20 | - | - | - | - | |
| Valeric-d9-Acid | 5.44 | 245.1 | 202.1 | 14 | 20 | - | - | - | - |
Retention time, fragmentation parameters of SCFA and range of concentrations of the calibration curve
Data Analysis
Analysis are validated only if Low, Middle and High QC at the begginning and at the end of the sequences have a CV < 15 %
Software
MassLynx
NAME
Waters
DEVELOPER
A.3. Quantification of Tryptophan (TRP) intermediates
6h
Internal Standards Solutions
Citation
LINK
The internal standards required are: Quinolinic-d3-acidToronto Research Chemicals Inc ,3-hydroxyanthranilic-d3-acidToronto Research Chemicals Inc , 5-hydroxyindole-3acetic-d5-acidToronto Research Chemicals Inc , melatonin d4Toronto Research Chemicals Inc , picolinic-d3-acidToronto Research Chemicals Inc , 3-hydroxykynurenine 13C2,15N1Toronto Research Chemicals Inc , L-kynurenine d4Toronto Research Chemicals Inc , indoleacetic-d4-acidToronto Research Chemicals Inc , kynurenic-d5-acidToronto Research Chemicals Inc , tryptamine d4Toronto Research Chemicals Inc , tryptophaneMerck MilliporeSigma (Sigma-Aldrich) , L-tryptophan d5Toronto Research Chemicals Inc , serotonin d4Toronto Research Chemicals Inc , indoxyl-13C6 sulfate potassium saltToronto Research Chemicals Inc , 5-hydroxy-L-tryptophan d4Toronto Research Chemicals Inc , 1H-indole-d5-3-acetamideToronto Research Chemicals Inc , tryptophol d4Toronto Research Chemicals Inc , N-acetylserotonine d3Toronto Research Chemicals Inc , indole-3-aldehyde 13C8Toronto Research Chemicals Inc & 3-indolepropionic-d2-acidToronto Research Chemicals Inc
Mix in one 15 mL falcon tube all the following to create the solution "SEI" :
| A | B | C | |
| Internal Standard | Volume SM0 (µL) | Volume SM1 (µL) | |
| Quinolinic-d3-acid | 17 | ||
| 3-hydroxyanthranilic-d3-acid | 31 | ||
| 5-hydroxyindole-3acetic-d5-acid | 118 | ||
| melatonin d4 | 94 | ||
| picolinic-d3-acid | 10 | ||
| 3-hydroxykynurenine 13C2,15N1 | 26 | ||
| L-kynurenine d4 | 65 | ||
| indoleacetic-d4-acid | 14 | ||
| kynurenic-d5-acid | 2.3 | ||
| tryptamine d4 | 2.4 | ||
| xanthurenic-d4-acid | 2.5 | ||
| L-tryptophan d5 | 335 | ||
| serotonin d4 | 86 | ||
| indoxyl-13C6 sulfate potassium salt | 51 | ||
| 5-hydroxy-L-tryptophan d4 | 134 | ||
| 1H-indole-d5-3-acetamide | 54 | ||
| tryptophol d4 | 2.6 | ||
| indole-3-lactic-d5-acid | 13 | ||
| N-acetylserotonine d3 | 177 | ||
| indole-3-aldehyde 13C8 | 6.1 | ||
| 3-indolepropionic-d2-acid | 3.8 | ||
| Water to complete to 10 mL (µL) | 8753 |
Homogeneized and then prepared a dilution by 40 in a 20 mL Flacon, with Methanol HiPerSolv CHROMANORMVWR International (Avantor) : this solution is call SEI_40
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Standards Solutions & Calibration Curve
The standards required are : picolinic acidMerck MilliporeSigma (Sigma-Aldrich) , 3-hydroxykynurenineMerck MilliporeSigma (Sigma-Aldrich) , quinolinic acidMerck MilliporeSigma (Sigma-Aldrich) , serotoninMerck MilliporeSigma (Sigma-Aldrich) , 5-hydroxytryptophaneMerck MilliporeSigma (Sigma-Aldrich) , kynurenineMerck MilliporeSigma (Sigma-Aldrich) , 3-hydroxyanthranilic acidMerck MilliporeSigma (Sigma-Aldrich) , tryptamineMerck MilliporeSigma (Sigma-Aldrich) , tryptophaneMerck MilliporeSigma (Sigma-Aldrich) , 5-hydroxyindole acetic acidToronto Research Chemicals Inc , indoxyl-3-sulfateToronto Research Chemicals Inc , N-acetylserotonineMerck MilliporeSigma (Sigma-Aldrich) , xanthurenic acidMerck MilliporeSigma (Sigma-Aldrich) , indole-3-acetamideMerck MilliporeSigma (Sigma-Aldrich) , kynurenic acidMerck MilliporeSigma (Sigma-Aldrich) , indole-3-lactic acidMerck MilliporeSigma (Sigma-Aldrich) , indole-3-aldehydeMerck MilliporeSigma (Sigma-Aldrich) , tryptopholMerck MilliporeSigma (Sigma-Aldrich) , indole-3-acetic acidMerck MilliporeSigma (Sigma-Aldrich) , melatonineMerck MilliporeSigma (Sigma-Aldrich) , indole-3-propionic acidMerck MilliporeSigma (Sigma-Aldrich)
Mix in one 4 mL glass tube all the following :
| A | B | C | |
| Metabolite | Volume SM0 (µL) | Volume SM1 (µL) | |
| picolinic acid | 17 | ||
| 3-hydroxykynurenine | 31 | ||
| quinolinic acid | 118 | ||
| serotonin | 94 | ||
| 5-hydroxytryptophane | 10 | ||
| kynurenine | 26 | ||
| 3-hydroxyanthranilic acid | 65 | ||
| tryptamine | 14 | ||
| tryptophane | 2.3 | ||
| 5-hydroxyindole acetic acid | 2.4 | ||
| indoxyl-3-sulfate | 2.5 | ||
| N-acetylserotonine | 335 | ||
| xanthurenic acid | 86 | ||
| indole-3-acetamide | 51 | ||
| kynurenic acid | 134 | ||
| indole-3-lactic acid | 54 | ||
| indole-3-aldehyde | 2.6 | ||
| tryptophol | 13 | ||
| indole-3-acetic acid | 177 | ||
| melatonine | 6.1 | ||
| indole-3-propionic acid | 3.8 | ||
| Water to complete to 2 mL (µL) | 877 |
This solution is called SG0
Dilute SGO by 100 to created SG1
Prepare a solution of BSA :
- 400 mg BSA
- 10 mL PBS
- Planar agitation for 00:20:00 , strength 8/10 at Room temperature
- Transfert in another falcon to remove any foalm
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
20m
Calibration Curve
Calibration Curve:
| A | B | C | D | E | F | G | H | |
| From which ? | SG1 | SG1 | SG1 | SG0 | SG0 | SG0 | SG0 | |
| Calibration Curve Solutions | L1 | L2 | L3 | L4 | L5 | L6 | L7 | |
| V(SG), µL | 75 | 250 | 500 | 25 | 75 | 250 | 500 | |
| V(H2O), µL | 425 | 250 | 0 | 475 | 425 | 250 | 0 | |
| V(BSA) (µL) | 500 | 500 | 500 | 500 | 500 | 500 | 500 |
Preparation of calibration curve solutions (a solution si named Lx; x from 1 (less concentrated) to 7 (most concentrated))
| A | B | C | D | |
| Solution | QC Low | QC Middle | QC High | |
| V(SG), µL | 20 | 200 | 400 | |
| V(H2O), µL | 480 | 300 | 100 | |
| V(BSA) (µL) | 500 | 500 | 500 |
Preparation of calibration curve QC, all from SG0
Extraction of DBS
In the whole DBS card Whatman 903TM, retrieved 2 punches (1 punch = 3 mm disc) with a puncher
WARNINGS:
- a punch must be entirely soaked in blood
- if possible, do not punch near the exterior of the blood droplets of the DBS
Put the 2 punches in a 1.5 mL Eppendorf vial
Add 200 µL of SEI_40
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
00:30:00 at -20 °C
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 175 µL of supernateant in a 2 mL glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Evaporate: 01:00:00 , 40 °C , vacuum
Add 100 µL of Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:9)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 85 µL in a glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Extraction of Serum
Make aliquots of 50 µL of serum
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Add 200 µL of SEI_40
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
00:30:00 at -20 °C
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 175 µL of supernateant in a 2 mL glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Evaporate: 01:00:00 , 40 °C , vacuum
Add 100 µL of Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:9)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 85 µL in a glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
LC parameters
Phase A:
- Water milliQMerck MilliporeSigma (Sigma-Aldrich) + 0.5% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Phase B:
- Methanol HiPerSolv CHROMANORMVWR International (Avantor) + 0.5% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Chromatographic System
Equipment
Acquity I-Class UPLC
NAME
Chromatography
TYPE
Waters
BRAND
Install the column in the oven
Equipment
Kinetex 1.7µm XB-C18 100A
NAME
Column
TYPE
Phenomenex
BRAND
00F-4498-AN
SKU
LC Column 150 x 2.1mm
SPECIFICATIONS
Adjust the temperature (55 °C ) and let it heat the core of the column
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Purge the system:
- 1 time at 100%A and 1 time at 100%B with open valve
- 2 times with open valve, 2 times with close valve at 50%A and 50%B
- Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
- Check that the pression is stable; otherwise : purge again
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 2%B
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0 | 0.4 | 5 | |
| 0 | 0.4 | 5 | |
| 2 | 0.4 | 10 | |
| 4 | 0.4 | 15 | |
| 6 | 0.4 | 30 | |
| 8 | 0.4 | 40 | |
| 9 | 0.4 | 45 | |
| 9.5 | 0.4 | 100 | |
| 11 | 0.4 | 00 | |
| 11.5 | 0.4 | 5 | |
| 13.5 | 0.4 | 5 |
Injection volume : 5 µL for both samples
Mass Spectrometry
Mass Spectrometer
Equipment
Xevo TQ-XS, with an ESI probe
NAME
Mass Spectrometer
TYPE
Waters
BRAND
Data Analysis
Analysis are validated only if Low, Middle and High QC at the begginning and at the end of the sequences have a CV < 15 %
Software
MassLynx
NAME
Waters
DEVELOPER
A.4. Quantification of Bile Acids
6h
Internal Standards (IS) Solutions
- The internal standards required are: cholic acid d5Toronto Research Chemicals Inc ,cholic acid sulfate d4Toronto Research Chemicals Inc , glycocholic acid d4Toronto Research Chemicals Inc , taurocholic acid d4Toronto Research Chemicals Inc , ursodeoxycholic acid d4Toronto Research Chemicals Inc ,chenodeoxycholic acid d4Toronto Research Chemicals Inc , deoxycholic acid d5Toronto Research Chemicals Inc , lithocholic acid d4Toronto Research Chemicals Inc
- Prepared solution of 1 mg/mL of each IS, called SM
Mix in one 10 mL Falcon tube all the following to create the solution "SEI" :
| A | B | |
| Internal Standard | Volume SM (µL) | |
| Cholic acid d5 | 4.1 | |
| cholic acid sulfate d4 | 5.4 | |
| glycocholic acid d4 | 9.4 | |
| taurocholic acid d4 | 10.8 | |
| ursodeoxycholic acid d4 | 2.0 | |
| chenodeoxycholic acid d4 | 2.0 | |
| deoxycholic acid d5 | 1.6 | |
| lithocholic acid d4 | 19.0 | |
| Methanol/water (1:1) to complete to 10 mL (µL) | 4946 |
Homogeneized and then prepared 32 mL of a dilution by 40 in a 20 mL Flacon, with Isopropanol HyperSolv CHROMANORMVWR International (Avantor) : this solution is call SEI_40
Standards Solutions & Calibration Curve
- The standards required are : taurohyocholic acidMerck MilliporeSigma (Sigma-Aldrich) ,glycohyocholic acidMerck MilliporeSigma (Sigma-Aldrich) ,glycodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich) ,lithocholic acidMerck MilliporeSigma (Sigma-Aldrich) , chenodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich) ,glycolithcholic acidMerck MilliporeSigma (Sigma-Aldrich) ,glycocholic acidMerck MilliporeSigma (Sigma-Aldrich) , cholic acid sulfateMerck MilliporeSigma (Sigma-Aldrich) ,taurochenodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich) ,ursodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich) , glycochenodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich) ,taurodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich) ,lithocholic acid sulfateMerck MilliporeSigma (Sigma-Aldrich) ,taurocholic acidMerck MilliporeSigma (Sigma-Aldrich) ,taurolithocholic acidMerck MilliporeSigma (Sigma-Aldrich) ,hyocholic acidMerck MilliporeSigma (Sigma-Aldrich) ,glycoursodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich) ,tauroursodeoxycholic acidMerck MilliporeSigma (Sigma-Aldrich) ,cholic acidMerck MilliporeSigma (Sigma-Aldrich) ,deoxycholic acidMerck MilliporeSigma (Sigma-Aldrich)
- Prepare standard solution of 1 mg/mL for each standard and a diluted one, by 100, for glycolithocholic acid, urosdeoxycholic acid, taurolithocholic acid and hyocholic acid
Mix in one 4 mL glass tube all the following :
| A | B | C | |
| Metabolite | Volume SM0 (µL) | Volume SM1 (µL) | |
| taurohyocholic acid | 2.2 | ||
| glycohyocholic acid | 1.9 | ||
| glycodeoxycholic acid | 4.5 | ||
| lithocholic acid | 38 | ||
| chenodeoxycholic acid | 3.9 | ||
| glycolithcholic acid | 173 | ||
| glycocholic acid | 9.0 | ||
| cholic acid sulfate | 2.1 | ||
| taurochenodeoxycholic acid | 5.2 | ||
| ursodeoxycholic acid | 157 | ||
| glycochenodeoxycholic acid | 13.5 | ||
| lithocholic acid sulfate | 2.0 | ||
| taurodeoxycholic acid | 5.2 | ||
| taurocholic acid | 5.4 | ||
| taurolithocholic acid | 193 | ||
| hyocholic acid | 163 | ||
| glycoursodeoxycholic acid | 4.5 | ||
| tauroursodeoxycholic acid | 5.0 | ||
| cholic acid | 4.1 | ||
| deoxycholic acid | 3.9 | ||
| Water to complete to 2 mL (µL) | 1202 |
This solution is called SG0
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Dilute SGO by 100 to created SG1
Prepare a solution of BSA :
- 400 mg BSA
- 10 mL PBS
- Planar agitation for 00:20:00 , strength 8/10 at Room temperature
- Transfert in another falcon to remove any foalm
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Calibration Curve
Calibration Curve:
| A | B | C | D | E | F | G | H | |
| From which ? | SG1 | SG1 | SG1 | SG0 | SG0 | SG0 | SG0 | |
| Calibration Curve Solutions | L1 | L2 | L3 | L4 | L5 | L6 | L7 | |
| V(SG), µL | 75 | 250 | 500 | 25 | 75 | 250 | 500 | |
| V(H2O), µL | 425 | 250 | 0 | 475 | 425 | 250 | 0 | |
| V(BSA) (µL) | 500 | 500 | 500 | 500 | 500 | 500 | 500 |
Preparation of calibration curve solutions (a solution si named Lx; x from 1 (less concentrated) to 7 (most concentrated))
| A | B | C | D | |
| Solution | QC Low | QC Middle | QC High | |
| V(SG), µL | 20 | 200 | 400 | |
| V(H2O), µL | 480 | 300 | 100 | |
| V(BSA) (µL) | 500 | 500 | 500 |
Preparation of calibration curve QC, all from SG0
Extraction of DBS
In the whole DBS card Whatman 903TM, retrieved 2 punches (1 punch = 3 mm disc) with a puncher
WARNINGS:
- a punch must be entirely soaked in blood
- if possible, do not punch near the exterior of the blood droplets of the DBS
Put the 2 punches in a 1.5 mL Eppendorf vial
Add 400 µL of SEI_40
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
00:30:00 at -20 °C
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 350 µL of supernateant in a 2 mL glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Evaporate: 01:00:00 , 40 °C , vacuum
Add 100 µL of Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:1)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 85 µL in a glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Extraction of Serum
Make aliquots of 50 µL of serum
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Add 400 µL of SEI_40
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
00:30:00 at -20 °C
Transfert 375 µL of supernateant in a 2 mL glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Evaporate: 01:00:00 , 40 °C , vacuum
Add 100 µL of Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:1)
Vigourously vortexed during 00:01:00
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 85 µL in a glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
LC parameters
Phase A:
- Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (9:1) + 1 mM formiate ammoniumVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Phase B:
- Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Isopropanol HyperSolv CHROMANORMVWR International (Avantor) (1:1)
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Chromatographic System
Equipment
Acquity I-Class UPLC
NAME
Chromatography
TYPE
Waters
BRAND
Install the column in the oven
Adjust the temperature (60 °C ) and let it heat the core of the column
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Purge the system:
- 1 time at 100%A and 1 time at 100%B with open valve
- 2 times with open valve, 2 times with close valve at 50%A and 50%B
- Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
- Check that the pression is stable; otherwise : purge again
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 2%B
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0.0 | 0.6 | 10 | |
| 0.0 | 0.6 | 10 | |
| 0.1 | 0.6 | 10 | |
| 9.25 | 0.6 | 35 | |
| 11.5 | 0.65 | 85 | |
| 11.8 | 0.8 | 100 | |
| 12.0 | 1 | 100 | |
| 12.1 | 1 | 100 | |
| 12.4 | 0.85 | 100 | |
| 12.45 | 0.85 | 55 | |
| 12.5 | 0.8 | 10 | |
| 12.6 | 0.7 | 10 | |
| 12.7 | 0.6 | 10 | |
| 15.0 | 0.6 | 10 |
Citation
LINK
Injection volume : 5 µL for both samples
Mass Spectrometry
Mass Spectrometer
Equipment
Xevo TQ-XS, with an ESI probe
NAME
Mass Spectrometer
TYPE
Waters
BRAND
Data Analysis
Analysis are validated only if Low, Middle and High QC at the begginning and at the end of the sequences have a CV < 15 %
Software
MassLynx
NAME
Waters
DEVELOPER
A.5. Quantification of TriCarboxylic Acids intermediates
6h
Internal Standards (IS) Solutions
- The internal standards required are:
- Prepare a solution of 1 mg/mL of each IS, called SM
Mix in one 10 mL Falcon tube all the following to create the solution "SEI" :
| A | B | |
| Internal Standard | Volume SM (µL) | |
| Succinic acid 13C4 | 4 | |
| Fumaric acid 13C4 | 8 | |
| Malic acid 13C4 | 4 | |
| Itaconic acid 13C4 | 2 | |
| Citric acid 13C4 | 10 | |
| Lactic acid 13C2 | 4 | |
| V(water) to complete to 2 mL (µL) | 1978 |
Homogeneized and then prepared 40 mL of a dilution by 40 in a 50 mL Flacon, with Water milliQMerck MilliporeSigma (Sigma-Aldrich) : this solution is call SEI_40
Standards Solutions & Calibration Curve
- The standards required are: citric acidMerck MilliporeSigma (Sigma-Aldrich) ,isocitric acidMerck MilliporeSigma (Sigma-Aldrich) ,aconitic acidMerck MilliporeSigma (Sigma-Aldrich) , ketoglutaric acidMerck MilliporeSigma (Sigma-Aldrich) ,succinic acidMerck MilliporeSigma (Sigma-Aldrich) , fumaric acidMerck MilliporeSigma (Sigma-Aldrich) ,malic acidMerck MilliporeSigma (Sigma-Aldrich) , itaconic acidMerck MilliporeSigma (Sigma-Aldrich) , lactic acidMerck MilliporeSigma (Sigma-Aldrich)
- Diluted one, by 100, for glycolithocholic acid, urosdeoxycholic acid, taurolithocholic acid and hyocholic acid
Mix in one 4 mL glass tube all the following :
| A | B | |
| Metabolite | Volume SM0 (µL) | |
| Citric acid | 115 | |
| Isocitric acid | 115 | |
| Aconitic acid | 35 | |
| Ketoglutaric acid | 29 | |
| Succinic acid | 24 | |
| Fumaric acid | 2 | |
| Malic acid | 27 | |
| Itaconic acid | 3 | |
| Lactic acid | 144 | |
| Water to complete to 2 mL (µL) | 1505 |
This solution is called SG0
Dilute SGO by 100 to created SG1
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Calibration Curve
Calibration Curve:
| A | B | C | D | E | F | G | H | |
| From which ? | SG1 | SG1 | SG1 | SG0 | SG0 | SG0 | SG0 | |
| Calibration Curve Solutions | L1 | L2 | L3 | L4 | L5 | L6 | L7 | |
| V(SG), µL | 75 | 250 | 500 | 25 | 75 | 250 | 500 | |
| V(H2O), µL | 925 | 750 | 500 | 975 | 925 | 750 | 500 |
Preparation of calibration curve solutions (a solution si named Lx; x from 1 (less concentrated) to 7 (most concentrated))
| A | B | C | D | |
| Solution | QC Low | QC Middle | QC High | |
| V(SG), µL | 20 | 200 | 400 | |
| V(H2O), µL | 980 | 800 | 600 |
Preparation of calibration curve QC, all from SG0
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Extraction of DBS
In the whole DBS card Whatman 903TM, retrieved 2 punches (1 punch = 3 mm disc) with a puncher
WARNINGS:
- a punch must be entirely soaked in blood
- if possible, do not punch near the exterior of the blood droplets of the DBS
Put the 2 punches in a 1.5 mL Eppendorf vial
Add 200 µL of SEI_40
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
00:30:00 at -20 °C
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 175 µL of supernateant in a 2 mL glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Evaporate: 01:00:00 , 40 °C , vacuum
Add 100 µL of Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:9)
Vigourously vortexed during 00:01:00
10000 x g, 4°C, 00:10:00
Transfert 85 µL in a glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Extraction of Serum
Make aliquots of 50 µL of serum
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Add 200 µL of SEI_40
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
00:30:00 at -20 °C
Transfert 175 µL of supernateant in a 2 mL glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Evaporate: 01:00:00 , 40 °C , vacuum
Add 100 µL of Methanol HiPerSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (1:9)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 85 µL in a glass vial
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
LC parameters
Phase A:
- Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Phase B:
- Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) + 0.1% Formic acid >99% for LC-MS VWR International (Avantor)Catalog #84865.260
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Chromatographic System
Equipment
Acquity I-Class UPLC
NAME
Chromatography
TYPE
Waters
BRAND
Install the column in the oven
Equipment
Kinetex 1.7µm XB-C18 100A
NAME
Column
TYPE
Phenomenex
BRAND
00F-4498-AN
SKU
LC Column 150 x 2.1mm
SPECIFICATIONS
Adjust the temperature (55 °C ) and let it heat the core of the column
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Purge the system:
- 1 time at 100%A and 1 time at 100%B with open valve
- 2 times with open valve, 2 times with close valve at 50%A and 50%B
- Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
- Check that the pression is stable; otherwise : purge again
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 2%B
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0.0 | 0.6 | 10 | |
| 0.0 | 0.6 | 10 | |
| 0.1 | 0.6 | 10 | |
| 9.25 | 0.6 | 35 | |
| 11.5 | 0.65 | 85 | |
| 11.8 | 0.8 | 100 | |
| 12.0 | 1 | 100 | |
| 12.1 | 1 | 100 | |
| 12.4 | 0.85 | 100 | |
| 12.45 | 0.85 | 55 | |
| 12.5 | 0.8 | 10 | |
| 12.6 | 0.7 | 10 | |
| 12.7 | 0.6 | 10 | |
| 15.0 | 0.6 | 10 |
Citation
LINK
Injection volume : 5 µL for both samples
Mass Spectrometry
Mass Spectrometer
Equipment
Xevo TQ-XS, with an ESI probe
NAME
Mass Spectrometer
TYPE
Waters
BRAND
Data Analysis
Analysis are validated only if Low, Middle and High QC at the begginning and at the end of the sequences have a CV < 15 %
Software
MassLynx
NAME
Waters
DEVELOPER
B.1. Untargeted Lipidomics
8h
Extraction of DBS
In the whole DBS card Whatman 903TM, retrieved 2 punches (1 punch = 3 mm disc) with a puncher
WARNINGS:
- a punch must be entirely soaked in blood
- if possible, do not punch near the exterior of the blood droplets of the DBS
Put the 2 punches in a 1.5 mL Eppendorf vial
Add 1000 µL of Isopropanol HyperSolv CHROMANORMVWR International (Avantor)
Vigourously vortexed during 00:01:00
Planar agitation : 01:00:00 ,Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 900 µL of supernateant in a glass vial of 1.5 mL
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Dry for 02:00:00 under vacuum at 40 °C
2h
Dried extract is redissolved with 100 µL of:
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Isopropanol HyperSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (6:3:1)
Vigorously vortexed during 00:01:00 at Room temperature
Transfert whole aliquot in a 200 µL 96-well plates
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Extraction of Serum
Make aliquots of 50 µL of serum
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Add 950 µL of cold Isopropanol HyperSolv CHROMANORMVWR International (Avantor) (-20 °C )
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
Let it 00:20:00 at -20 °C
Planar agitation : 01:00:00 ,Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 900 µL of supernateant in a glass vial of 1.5 mL
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Dry for 02:00:00 under vacuum at 40 °C
2h
Dried extract is redissolved with 100 µL of:
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Isopropanol HyperSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (6:3:1)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigorously vortexed during 00:01:00 at Room temperature
Transfert whole aliquot in a 100 µL 96-well plates
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
LC parameters
Phase A:
- 200 mL + 4.0 mL * [Number of injections]
- Water milliQMerck MilliporeSigma (Sigma-Aldrich) /Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Isopropanol HyperSolv CHROMANORMVWR International (Avantor) (5:3:2)
- 1 mM of formiate ammoniumVWR International (Avantor)
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Phase B:
- 200 mL + 4.0 mL * [Number of injections]
- Water milliQMerck MilliporeSigma (Sigma-Aldrich) /Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Isopropanol HyperSolv CHROMANORMVWR International (Avantor) (1:9:90)
- 10 mM of formiate ammoniumVWR International (Avantor)
- Stir vigorously during 00:01:00 by hand
- Ultrasound during 00:10:00 to remove air bubble
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Chromatographic System
Equipment
Ultimate 3000
NAME
UHPLC
TYPE
Dionex Softron
BRAND
SRD-3400
SKU
Pump, Column Compartment, AutoSampler
SPECIFICATIONS
Install the column in the oven
Equipment
ACQUITY UPLC BEH C18 (2.1x100x1.7)
NAME
Column
TYPE
Acquity
BRAND
Adjust the temperature (60 °C ) and let it heat the core of the column
Prepare the cleaning solvents (same solvent as the recovery solvent) & ultrasound it during 00:05:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Prepare the piston “lubrication” solution (90% Methanol HiPerSolv CHROMANORMVWR International (Avantor) and 10%Water milliQMerck MilliporeSigma (Sigma-Aldrich) ) & ultrasound it during 00:10:00 to remove air bubbles
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
Purge the system:
- 1 time at 100%A and 1 time at 100%B with open valve
- 2 times with open valve, 2 times with close valve at 50%A and 50%B
- Set the flow at 2 mL/min at 50%A and 50%B during 00:10:00
- Check that the pression is stable; otherwise : purge again
Balancing the system: Gradually reach the flow rate of 0.5 mL/min at 2%B
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0 | 0.4 | 10 | |
| 0 | 0.4 | 10 | |
| 2.7 | 0.4 | 45 | |
| 2.8 | 0.4 | 53 | |
| 8 | 0.4 | 60 | |
| 8.1 | 0.4 | 80 | |
| 11.5 | 0.4 | 80 | |
| 12 | 0.4 | 100 | |
| 13 | 0.4 | 100 | |
| 13.2 | 0.4 | 10 |
Volume of injection (µL): 5 µL for both samples
Mass Spectrometry
Mass Spectrometer
Equipment
Q-Exactive
NAME
Mass Spectrometer
TYPE
ThermoScientific
BRAND
HESI Source
SPECIFICATIONS
MS Parameters :
- Scan Range: 250-1500 m/z
- Resolution MS1: 70,000
- Resolution MS2: 15,000
- Polarity: POS or NEG
- Microscans: 1
- Lock mass: None
- AGC Target: 1E6
- Maximum Inject Time: 200
- Sheath Gas Flow Rate: 45
- Aux Gas Flow rate: 10
- Sweep Gas flow Rate: 1
- Spray Voltage (kV): +/-3.5
- Capillary Temperature (°C): 250
- S-lens RF level: 25
- Aux gas Heater temperature (°C): 350
Cleaning the source:
- Set source temperature to 50 °C
- Let it "cool" 01:00:00
- Unplug the Ion MAx API source with HESI probe attached
- Unscrewed the Sweep Cone and the capillary
- Immediately plug another cleaned capillary
- Clean the sweep cone with wet Aluminium oxide powerMerck MilliporeSigma (Sigma-Aldrich)
- Rinse thoroughly with Water milliQMerck MilliporeSigma (Sigma-Aldrich)
- In a glass beaker, cover completely the Sweep Cone and the capillary with Methanol HiPerSolv CHROMANORMVWR International (Avantor)
- Ultrasound during 00:10:00
Equipment
Ultrasonic Cleaner USC - TH
NAME
Ultrasound bath
TYPE
VWR
BRAND
142-0084
SKU
230V 50Hz 370VA
SPECIFICATIONS
- Let it dry
- Replace the cone and the Ion Max API source with HESI probe
- Set source temperature back to normal
Reinstall the source & allow it to return to analysis conditions
Calibration:
- POS: Calibration Solution Pierce LTQ Velos ESI Positive IonThermo Fisher
- NEG: Calibration Solution Pierce ESI Negative IonThermo Fisher
- RMS < 0.4/0.4 in both ionisation mode
Equipment
Fusion 100T
NAME
syringe pump
TYPE
Chemyx Inc.
BRAND
Once calibrated, sequences can be run
Typical Sequences:
| Sample Type | Injection Volume (µL) | |
| Equilibrium(=sample) * 10 | 5 | |
| Blank Solvent | 5 | |
| Performance Evaluation * 3 | 5 | |
| Blank Solvent | 5 | |
| Sample QC | 5 | |
| Sample *10 | 5 | |
| Sample QC | 5 | |
| ... | 5 | |
| Last Sample QC | 5 | |
| Blank Solvent * 3 | 5 |
Data Analysis
Lipids Identification :
Software
LipidSearch
NAME
ThermoScientific
DEVELOPER
Features detection:
- Peak Picking on all samples is done by using Workflow4Metabolomics
- Automatic attribution of the precedent list
Software
Workflow4Metabolomics
NAME
doi: 10.1093/bioinformatics/btu813
DEVELOPER
REPOSITORY
SOURCE LINK
Fusion of modalites = sorting redundancies:
- Only the best modality is kept for 1 metabolite, based on RT > Dead Volume (0-1 min) and/or lower CV(metabolite) on QCs
B.2. Quantitative Lipidomics
6h
- Method from the paper :
Citation
LINK
- More details on internal standards used and quantification process on : https://doi.org/10.5281/zenodo.17184000
Extraction of DBS
In the whole DBS card Whatman 903TM, retrieved 2 punches (1 punch = 3 mm disc) with a puncher
WARNINGS:
- a punch must be entirely soaked in blood
- if possible, do not punch near the exterior of the blood droplets of the DBS
Put the 2 punches in a 1.5 mL Eppendorf vial
Add 1000 µL of Isopropanol HyperSolv CHROMANORMVWR International (Avantor) internal solutions
Vigourously vortexed during 00:01:00
Planar agitation : 01:00:00 ,Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 900 µL of supernateant in a glass vial of 1.5 mL
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Dry for 02:00:00 under vacuum at 40 °C
Dried extract is redissolved with 100 µL of:
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Isopropanol HyperSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (6:3:1)
Vigorously vortexed during 00:01:00 at Room temperature
Transfert whole aliquot in a 200 µL 96-well plates
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Extraction of Serum
Make aliquots of 50 µL of serum
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Add 950 µL of cold Isopropanol HyperSolv CHROMANORMVWR International (Avantor) of internal solutions
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigourously vortexed during 00:01:00
Let it 00:20:00 at -20 °C
Planar agitation : 01:00:00 ,Room temperature , strength 8/10
Equipment
Titramax 1000
NAME
Shaker
TYPE
Heidolph
BRAND
10000 x g, 4°C, 00:10:00
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
Transfert 900 µL of supernateant in a glass vial of 1.5 mL
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Dry for 02:00:00 under vacuum at 40 °C
Dried extract is redissolved with 100 µL of:
Acetonitrile HiPerSolv CHROMANORMVWR International (Avantor) /Isopropanol HyperSolv CHROMANORMVWR International (Avantor) /Water milliQMerck MilliporeSigma (Sigma-Aldrich) (6:3:1)
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
Vigorously vortexed during 00:01:00 at Room temperature
Transfert whole aliquot in a 100 µL 96-well plates
Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND
SFC parameters
Phase A:
- Supercritial CO2
Phase B:
- Water milliQMerck MilliporeSigma (Sigma-Aldrich) /Methanol HiPerSolv CHROMANORMVWR International (Avantor) (2:98)
- 20 mM of formiate ammoniumVWR International (Avantor)
Chromatographic System
Equipment
Ultra-Performance Convergence Chromatography (UPC²)
NAME
Chromatography
TYPE
Waters
BRAND
Install the column in the oven
Equipment
ACQUITY UPC² Torus DEA (100x3x1.7)
NAME
Column
TYPE
Acquity
BRAND
Adjust the temperature (40 °C ) and let it heat the core of the column
Gradient:
| Retention (min) | Flow (mL/min) | %B | |
| 0 | 1.2 | 1 | |
| 0 | 1.2 | 1 | |
| 0.5 | 1.2 | 1 | |
| 6 | 1.2 | 40 | |
| 6.1 | 1.2 | 65 | |
| 9.1 | 1.1 | 65 | |
| 12.1 | 1.2 | 1 |
Mass Spectrometry
Mass Spectrometer
Equipment
Xevo G2 XS QTof
NAME
Mass Spectrometer
TYPE
Waters
BRAND
MS Parameters :
- Resolution MS1: 16,000 at 400 m/z
- Mode: Full MS
- Scan Range: 50-1500 Da (centroïd)
- Polarity: POS or NEG
- Source Temperature (°C): 150
- Spray Voltage (kV): -2.6 & 3
- Desolvatation Gas Flow Rate (L/hr): 1000
- Cone Gas Flow Rate (L/hr): 50
- Desolvatation temperature (°C): 550
Data Analysis
Citations
Step 10.1
Monteiro, J. , Lefèvre, A. , Dufour-Rainfray, D. , Oury, A. , Chicheri, G. , Galineau, L. , Blasco, H. , Nadal-Desbarats, L. and Emond, P. Multi-Compartment SCFA Quantification in Human
10.4236/ajac.2024.156012Step 14
Lefèvre A, Mavel S, Nadal-Desbarats L, Galineau L, Attucci S, Dufour D, Sokol H, Emond. Validation of a global quantitative analysis ...
10.1016/j.talanta.2018.11.09404Step 27.10
Sarafian MH, Lewis MR, Pechlivanis A, Ralphs S, McPhail MJ, Patel VC, Dumas ME, Holmes E, Nicholson JK.. Bile acids profiling and quantification in ...
10.1021/acs.analchem.5b0155609/30/2015Step 35.10
Sarafian MH, Lewis MR, Pechlivanis A, Ralphs S, McPhail MJ, Patel VC, Dumas ME, Holmes E, Nicholson JK.. Bile acids profiling and quantification in ...
10.1021/acs.analchem.5b0155609/30/2015Step 39.2
Folch J, Lees M, Stanley. A simple method for the isolation ...
https://doi.org/10.1016/S0021-9258(18)64849-51957Step 43
Le Faouder, P.; Soullier, J.; Tremblay-Franco, M.; Tournadre, A.; Martin, J.-F.; Guitton, Y.; Carlé, C.; Caspar-Bauguil, S.; Denechaud, P.-D.; Bertrand-Michel, J. Untargeted Lipidomic Profiling of Dry Blood ...
https://doi.org/10.3390/metabo1105030501Step 45.2
Folch J, Lees M, Stanley. A simple method for the isolation ...
https://doi.org/10.1016/S0021-9258(18)64849-51957Step 46
Le Faouder, P.; Soullier, J.; Tremblay-Franco, M.; Tournadre, A.; Martin, J.-F.; Guitton, Y.; Carlé, C.; Caspar-Bauguil, S.; Denechaud, P.-D.; Bertrand-Michel, J. Untargeted Lipidomic Profiling of Dry Blood ...
https://doi.org/10.3390/metabo1105030501Acknowledgements
This work was supported by:
- the MetaboHUB infrastructure funded by the Agence Nationale de la Recherche under the France 2030 program (MetaboHUB ANR-11-INBS-0010 ; MetEx+ ANR-21-ESRE-0035; MetaboHUB (JVCE) ANR-24-INBS-0012)
- the Plateforme de Métabolomique et d’Analyses Chimiques, US-61, MetaboHUB-Tours
We would like to thank:
- the MetaboHUB-MetaToul-Lipidomique platform for the SFC analysis and for welcoming and training Jérémy Monteiro
- the CHRU of Tours for the help for sampling and the handling of samples