Non targeted Lipid Profiling LC-FT-MS/MS

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 Pump 2.2 Column 2.3 Autosampler 2.4 Mass spectrometer 3 Data analysis and quantitation 3.1 Data handling 3.2 Calibration and quantitation 4 Method validation 4.1 Precision

Sample preparation

• Lipid extracts are taken up in 100ul CHCl₃ / MeOH 1:1.

Material	Material used	Internal Standards	Internal Standard added
Tissue extracts	5 - 100 mg tissue	as needed	as needed

Instrumentation and method

Pump

• Type: Thermo Accela
• Mode: Gradient
• Solvent A: Water with 1% ammonia acetate and 0.1% formic acid
• Solvent B: Acetonitrile/2-propanol 5:2 with 1% ammonia acetate and 0.1% formic acid
• Gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	0.25	65	35
4.0	0.25	30	70
20.0	0.25	0	100
30.0	0.25	0	100

Column

• Type: Thermo Hypersil GOLD C18, 100x1 mm, 1.9µm
• Temperature: 50°C

Autosampler

• Type: Accela
• Injection volume: 5ul
• Wash solvent: Solvent B

Mass spectrometer

• Type: Thermo LTQ-FT Ultra
• Ionization mode: positive ESI
• Ionization voltage: 4500V
• Capillary temperature: 250°C
• Tube Lens: 120V
• Capillary voltage: 35V
• Damping gas: Helium
• Mass calibration: <2ppm external
• Mass resolution 200.000
• MS/MS-conditions:

Data Dependent Acquisition (DDA) in FT-preview mode

5 LTQ low resolution CID MS/MS spectra per cycle @ 35% energy

Repeat count: 2

Exclusion duration: 60s

Data analysis and quantitation

Data handling

• Peak areas for all analytes are calculated by Lipidomics^TM. Peak identification is based on exact mass (<2ppm) and retention time with database search. MS/MS data are analyzed for further structural conformation of data.

Calibration and quantitation

• calibration type: relative quantitation 
• The internal standard is set to 100% and all other peaks are expressed as % relative to internal standards.

Method validation

Precision

Method precision for 5 injections is better than 15%. Inter day retention time stability is better than 1%.