Cholesterol 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	20-50 ug

• Silylation of free OH containing molecules (sterols and fatty acids): 50 uL total lipid extract in chloroform-methanol=2:1 is evaporated, redissolved in 50 uL benzene, 5 uL BSA is added and the rection left at RT for an hour. A 1 µl aliquot is injected onto the BPX-1 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-1 (10m x 0.1mm x 0.1um)
  
• Temperature gradient

Rate	Final temperature	Hold time
-	150	0.5
30	290	3

• 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

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)