Fatty Acid Profiling GC-MS

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 GC  2.2 Column 2.3 Autosampler 2.4 Mass spectrometer 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

• 100µl of sample are spiked with internal standard. Following that samples are evaporated with nitrogen(g) and redisolved in 50µl of a PFBBr solution (3.4 % in acetonitrile) and 10µl of N,N-Diisopropyl ethanolamine. After 10min of incubation at RTsamples are evaporated under a gentle stream of nitrogen and resuspended in 50µl of hexane.
• sample sovlent: Hexane

Material	Material used	Internal Standard(s)	Internal Standard(s) added
Lipid extracts	100ul	15:0 FA	25ug

Instrumentation and method

GC 

• Type: Thermo Trace GC
• Mode: Split/Splitless
• Injector Temperature: 300°C
• Carrier gas: Helium
• Temperature gradient:

Time [min]	Flow [ml/min]	Temperature [°C]
0	1	150
1.0	1	150
9.0	1	200
19.0	1	325
21.0	1	325

Column

TR5MS 30m 

Autosampler

• Type: Thermo TriPlus
• Injection volume: 1µl
• Wash solvent: Hexane

Mass spectrometer

• Type: Thermo DSQ
• Ionization mode: NICI
• Scan type: Full Scan m/z 100-400
• Electron energy: 70eV
• Source temperature: 250°C
• CI gas: Methane
• Transfer Line: 330°C
• Mass table

Analyte	M-PFB [m/z]
12:0 FA	199
14:0 FA	227
14:1 FA	225
16:0 FA	255
16:1 FA	253
17:0 FA	269
18:0 FA	283
18:1n9 FA	281
18:1n11 FA	281
18:2 FA	279
18:3 FA	277
20:0 FA	311
20:1 FA	309
20:2 FA	307
20:3 FA	305
20:4 FA	303
20:5 FA	301
22:0 FA	339
22:6 FA	327
24:0 FA	367

Data analysis and quantification

Data handling

• Peak areas for the M-PFB anions are calculated by QuanBrowser, related to the internal standard peak area and expressed as percent of internal standard.

Calibration and quantification

• calibration type: relative quantitation
• Fatty acid peak areas are related to the internal standard peak area and expressed as percent of internal standard.

Method validation

Accuracy

Precision

Limit of detection

Recovery

Sample data

Mass spectra

Chromatogram

Reference

• PubMed
• electronic journal