Nov 19, 2024

Public workspaceTotal fatty acids extraction and analysis by GC-FID

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Protocol Citationjustine.bertrand-michel Bertrand-Michel 2024. Total fatty acids extraction and analysis by GC-FID . protocols.io https://protocols.io/view/total-fatty-acids-extraction-and-analysis-by-gc-fi-dfe93jh6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 11, 2024
Last Modified: November 19, 2024
Protocol Integer ID: 101569
Funders Acknowledgement:
Agence Nationale de la recherche
Grant ID: ANR-21-ESRE-0035
Abstract
Presentation of the extraction of total fatty acids (= TFA) from the tissue or fluid matrix for analysis by GC-FID TRACUS 1300.
A liquid-liquid extraction was realized following modified Bligh and Dyer protocol. A hydrolysis and a derivatization were performed. The extract was analyzed by gas chromatography with flame ionization detector.
A quantitative analysis of conventional fatty acids (c10:0, c12:0, c14:0, c15:0, c16:0, c17:0, c18:0, c20:0, c22:0,c23:0, c24:0, c14:1w5,c15:1,c16:1w7,c16:1w9,c18:1w9, c18:1w7, c20:1w9, c22:1w9, c24:1w9, c18:2w6, c18:3w6, c18:3w3,c18:3w4,c20:2w6, c20:3w3, c20:3w6, c20:4w6, c20:5w3, c22:2w6, c22:3w6, c22:5w3, c22:6w3, c22:4w6) with internal calibration (internal standard c19) was done.
Protocol materials
ReagentPotassium hydroxideHoneywell FlukaCatalog #P5958500G
Step 1.3
Reagent Methanol Merck MilliporeSigma (Sigma-Aldrich)
In 3 steps
ReagentAcetic acid solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #33209-M
Step 1.2
ReagentSupelco 37 components FAME mixMerck MilliporeSigma (Sigma-Aldrich)Catalog #CRM47885
Step 2.1
ReagentLysing Matrix A 2 mL tubeMP BiomedicalsCatalog #SKU 116910050-CF
Step 1.1
ReagentEGTAMerck MilliporeSigma (Sigma-Aldrich)
Step 1.1
Reagentglyceryl trinonadecanoateMerck MilliporeSigma (Sigma-Aldrich)Catalog #T4632-1G
Step 1.1
ReagentLysing matrix D tubesMP BiomedicalsCatalog #6913100
Step 1.1
ReagentHeptaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #32287-M
In 2 steps
ReagentBoron trifluoride-methanol solutionSupelcoCatalog #15716-1L
Step 1.3
ReagentEthyl acetateHoneywell FlukaCatalog #349722.5L
In 3 steps
ReagentDichloromethaneHoneywell FlukaCatalog #32222-2.5L
Step 1.2
Safety warnings
Wear appropriate clothing, closed shoes, lab coat, glasses, gloves, extractor hood
Use of CMR solvents, use of nitrogen
Before start
Verify the necessary quantity of samples, the availability of solvents and internal standard, the lab equipment necessary for extraction (lysing matrix, tubes, vials).
Ensure that samples are correctly identified to ensure traceability

Sample preparation
Sample preparation
1h 57m
1h 57m
Sample preparation
The preparation, extraction and derivatisation stages are explained in the following paragraph in 4 main parts:
1. Sample type
2. Bligh & dyer
3. Derivatisation
4. Injection preparation


Sample type

A.Tissues

1. Collect or weigh samples
2. Put samples in a lysing matrix
( ref : ReagentLysing Matrix A 2 mL tubeMP BiomedicalsCatalog #SKU 116910050-CF , ReagentLysing matrix D tubesMP BiomedicalsCatalog #6913100 )
3. Add 1 mL H2O(EGTA 5mM):MeOH (1:2) (ref: ReagentEGTAMerck MilliporeSigma (Sigma-Aldrich) Reagent Methanol Merck MilliporeSigma (Sigma-Aldrich) )
4. Crash samples until you obtain homogenat with a Fast prep : 6.5m/s, times : 2x30 sec
Equipment
FastPrep-24 5G
NAME
Bead beater
TYPE
MP Biomedicals
BRAND
116005500
SKU
LINK

5. Collect a equivalent volume depending of the type of sample
6. Add 10 µL of Internal standard TG 57:0 Reagentglyceryl trinonadecanoateMerck MilliporeSigma (Sigma-Aldrich)Catalog #T4632-1G corresponding at 4 µg

Liver : Amount1 mg type of the lysing matrix tube D
Lung : Amount1 mg type of the lysing matrix tube D
Muscles : Amount1 mg Type of the lysing matrix tube A

B. Fluid samples

1. Collect a equivalent volume depending of the type of sample (cf. below)
2. Add internal standard 10 µL equivalent at 4µg

Plasma : Amount10 µL
Lymphe : Amount20 µL


Bligh & dyer extraction

  1. In the pyrex tube : Mixture of solvant with sample and IS prepared in the previous step
  2. Add 2.5 mL MeOH, 2% acetic acid Reagent Methanol Merck MilliporeSigma (Sigma-Aldrich) ReagentAcetic acid solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #33209-M
  3. Add 2.5 mL of CH2Cl2 (ref : ReagentDichloromethaneHoneywell FlukaCatalog #32222-2.5L )
  4. Add 2 mL of H2O milliQ
  5. Mix sample during 2 min at RT
  6. Centrifuge sampleCentrifigation2500 rpm, 24°C, 00:06:00

  1. Aspirate the upper phase with a vacuum pump
  2. Recover the lower phase in new tubes
  3. Evaporate sample under nitrogen gas



6m

Derivatisation

Add 1 mL of MeOH with KOH 0.5M ReagentPotassium hydroxideHoneywell FlukaCatalog #P5958500G
  1. Mix sample during 2 min at RT
  2. Heat the tube : Temperature55 °C Duration00:30:00
  3. Cool down Duration00:10:00
  4. Add 1mL of heptane (ref :ReagentHeptaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #32287-M )
  5. Add 1 mL of BF3/MeOH ReagentBoron trifluoride-methanol solutionSupelcoCatalog #15716-1L Reagent Methanol Merck MilliporeSigma (Sigma-Aldrich)
  6. Mix sample during 2 min at RT
  7. Heat the tube : Temperature80 °C Duration01:00:00
  8. Cool down Duration00:10:00
  9. Add 1 mL H2O
  10. Add 2 mL of Heptane ReagentHeptaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #32287-M
  11. Mix sample during 2 min at RT
  12. Centrifuge : Centrifigation2500 rpm, 24°C, 00:01:00
  13. Take the upper phase with a pastor pipette and transfer in new tubes
  14. Dry sample under nitrogen gas





1h 51m

Injection preparation

  1. Transfer sample in vial with insert for the injection
  2. Add two times 80 µL of Ethyl Acetate (ReagentEthyl acetateHoneywell FlukaCatalog #349722.5L )
  3. Transfer of the 160 µl of Ethyl Acetate in vials with insert ReagentEthyl acetateHoneywell FlukaCatalog #349722.5L
  4. Dry under nitrogen gas
  5. Add 20 µL of Ethyl Acetate in vials ReagentEthyl acetateHoneywell FlukaCatalog #349722.5L


Calibration preparation
Calibration preparation
Absolute quantification of FAME
The calibration range is based on a mix of 37 fatty acids ( Ref :ReagentSupelco 37 components FAME mixMerck MilliporeSigma (Sigma-Aldrich)Catalog #CRM47885 ). We supplement the initial mix by adding 9 methylated fatty acids (C16 :2w6 ; C16 :2w4 ; C16 :4w3 ; C16 :3w3 ; C18 :1w7c ; C18 :4w3 ; C22 :3w3 ; C22 :5w3 ; C22 :4w6) at a concentration of 200ug/mL.

Preparation of cal 12 :

1. Add 50 µL of Supelco mix and 20 µL at 2 mg/mL of the fatty acids not present in the supelco
2. Evaporate and reconstitute in 100 µL of ethyl acetate

Preparation of the assay:

CAL 11 : 50 µL of CAL12 + 50 µL of ethyl acetate

CAL 10 : 50 µL of CAL11 + 50 µL of ethyl acetate

CAL 09 : 50 µL of CAL10 + 50 µL of ethyl acetate

CAL 08 : 50 µL of CAL09 + 50 µL of ethyl acetate

CAL 07 : 50 µL of CAL08 + 50 µL of ethyl acetate

CAL 06 : 50µL of CAL07 + 50 µL of ethyl acetate

CAL 05 : 50 µL of CAL06 + 50 µL of ethyl acetate

CAL 04 : 50 µL of CAL05 + 50 µL of ethyl acetate

CAL 03 : 50 µL of CAL04 + 50 µL of ethyl acetate

CAL 02 : 50 µL of CAL03 + 50 µL of ethyl acetate

CAL 01 : 50 µL of CAL02 + 50 µL of ethyl acetate

Add 22 µL of methylated C19 standard at 0.2 mg/mL in each cal

Evaporate and reconstitute to 50 µL of ethyl acetate.

Internal calibration/ External calibration

ISTD : TG19 or C19 at 0.2ug/uL :

1. Weigh 40 mg of C19 qsp 20mL of ethyl acetate.

2. Methylation of the C19 to be injected in GC-FID, see derivatisation extraction protocol.
Sample analysis
Sample analysis
Samples are analysed using a GC-FID system

Analytical conditions

--> Equipement :
Equipment
TRACE 1300
NAME
Gas Chromatograph
TYPE
Thermo Scientific
BRAND
43210167
SKU
coupled with a Flame Ionization Detector ( FID)
SPECIFICATIONS

--> Logiciel : Chroméléon 7.2

Injector conditions :

  1. Analytical conditions
  2. Volume d’injection : 1uL
  3. Température injecteur : 220°C
  4. Mode : Splitless
  5. Split flow : 60 mL/min
  6. Splitless time : 1 min
  7. Constant septum purge: ok
  8. Enable gas saver mode : ok
  9. Gas Saver Flow : 20 mL/min
  10. Gas saver time : 2 min
  11. Gaz vecteur : dihydrogène
  12. flow of the vector gaz : 1.5mL/min
  13. Rinsing solvent for injection needle and GC-FID system: ethyl acetate

Oven conditions :
Column : FAMEWAX, longueur 30m, ID 0.32mm, film 0.25µm (Restek réf : 12498)


Detector conditions :

Signal settings
* Detector active : ☒
* Acquisition: 0 – 45 min
* Data collection rate: 10 Hz
Detector settings
* Detector temperature : 230°C
* Flame : ☒
* Enable flameout retry : ☒
* Ignition threshold: 0,0 pA
* Peak width: standard
Gas settings
* Air flow control : ☒
* Air flow: 350 ml/min
* Hydrogen flow control : ☒
* Hydrogen flow: 35 ml/min


Sequence
Prepare a sequence on Chroméléon: DATA → Create → Sequence → Next → Next →

1. Select the instrumental method
2. Select the reprocessing method
3. The data reporting system → Next → Finish →
4. Save the sequence in the year/month folder



In the sequence, complete :

1. Name of sample
2. Position of sample
3. Start the sequence with 2 blanks, a supelco and 2 blanks
4. Inject a blank every 10 samples
5. Save the sequence
6. Check in the "Queue" that there is nothing pending.
7. Then run the sequence
8. The instrument starts by installing the analysis parameters.




Datas analysis
Datas analysis


Acceptance criteria

1. Instrument inspected, maintained and calibrated
2. Memory effect : FA not detected in whites (area < 0.010)
3. overall performance : Detection of internal standards for the determination of FAs in mixed standards and biological samples
4. Check the linearity of the ranges and the accuracy. Deselect points if necessary to obtain an R² < 0.985.

Reprocessing software
1.select "Data Processing Home" at the top
2. select "Chromatogram" alone or with "Interactive Results" and uncheck the rest.
3. use the tools to manually modify the areas on the top tabs.
-> The easiest way is to select 'Layout'.


* Full Size: allows full zooming out

* Autoscale Signal: refocuses peaks on the chromato

* Undo Zoom: returns to the previous zoom.

* In the injections section, with the whole list on the left, you can select several samples for comparison using the pushpin to the right of the injections or ctl. In the 'comparison' section, 'overlaid' and 'stacked' are used to display the chromatograms in the same or two different windows. Signal offset' and 'time offset' allow you to align them in terms of baseline and retention time.

* You can use just the mouse to add peaks and move the integration (in length or height) by positioning it on the baseline,

* Use the "delete" key to delete.

* You can also use the mouse to zoom in on an area by clicking and dragging.

* F4" to move to the next sample

* Shift + F4 to return to the previous sample

* Right-click and 'split peak' to split a double peak.

To make reprocessing easier, it is preferable to zoom in on a defined area (e.g. C16, C16:1w9 and C16:1w7).

Relative or absolute quantification

Relative quantification : [Area of the lipid]/[ISTD area]*quantity of ISTD
Absolute quantification thank to the calibration curve
Protocol references
E. G. Bligh and W. J. Dyer. 1959. A RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATION. Canadian Journal of Biochemistry and Physiology. 37(8): 911-917. https://doi.org/10.1139/o59-099