Fatty Acid analysis - GCMS

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 GCMS 3 Data analysis and quantification 3.1 Data handling 3.2 Calibration and quantification 4 Method validation 4.1 Accuracy 4.2 Precision 4.3 Limit of detection 4.4 Recovery 5 Sample data 5.1 Mass spectra 5.2 Chromatogram 6 Reference

Sample preparation

• Lipid extraction according to modified Folch

Material	Material used	Internal Standard(s)	Internal Standard(s) added
Cultured cells	106-107 cells	15:0 FA or di17:0 PC	20-50 ug

• Fatty acid methyl ester (FAME) formation: 50 uL total lipid extract in chloroform-methanol=2:1 is evaporated, 2 mL of 5 % acetyl chloride in MeOH is added and the mixture is kept at 80°C for 2 h. The resulting FAMEs are extracted with hexane, the solvent evaporated, and the residue redissolved in 50 µl benzene. A 1 µl aliquot is injected onto the BPX-70 capillary column.

Instrumentation and method

GCMS

• Type: Shimadzu GCMS-QP2010
  
• Autosampler: AOC-20i
• Injection volume: 0.5-2 uL
• Injection mode: Split
• Split ratio: 1/50-1/200
• Injector temperature: 240°C
• Carrier gas: Helium
• Column: BPX-70 (10m x 0.1mm x 0.2um)
  
• Temperature gradient

Rate	Final temperature	Hold time
-	150	2
6	215	0
15	235	6

• Ionization mode: EI
• Ionization voltage: 70 eV
  
• Source temperature: 200°C
  

Data analysis and quantification

Data handling

• peaks are identified and quantitated by using the GSMSSolution software with built-in NIST spectra database
  

Calibration and quantification

• quantitation type: relative (peak areas are related to the area of IS and expressed as weight percentage)
  

Method validation

Accuracy

Precision

Limit of detection

Sensitivity can be significantly enhanced in SIM mode when the MS detects only selected ions. The selection is made according to the base/main peaks in the MS spectrum of each FA class, i.e. saturated, monoene, etc.

Recovery

Sample data

Mass spectra

Chromatogram

Reference

• PubMed
• electronic journal

Please add categories as below for:

• analytical techniques applied
• lipid classes analyzed (see lipid class categories)