Sphingosine-1-phosphate - LC-MS/MS - Bielawski et al.

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 Pump 2.2 Autosampler 2.3 Column 2.4 Mass spectrometer 3 Data analysis and quantification 3.1 Software 3.2 Calibration and quantification 4 Method Validation 4.1 Accuracy 4.2 Precision 4.3 LOD 4.4 Recovery 5 Sample data 5.1 Mass spectra 5.2 Chromatogram 6 Reference

Sample preparation

• Sphingolipids were extracted with iso-propanol:water:ethyl acetate (30:10:60)
• The water phase is re-etxtracted
• 50 µL IS were added prior lipid extraction

Material	Material used	Internal Standard(s)	Internal Standard(s) added
Cells in culture	1 x e6 - 10 e6 cells	S1P 17:0, C17 SPH, 17C16 Cer, 18C17 Cer, 18C6-SM, 18C17-SM	1µM (S1P, SPH, Cer); 5µM SM
tissues	0.5 - 1 mg protein	S1P 17:0, C17 SPH, 17C16 Cer, 18C17 Cer, 18C6-SM, 18C17-SM	1µM (S1P, SPH, Cer); 5µM SM
serum	50 - 100µL	S1P 17:0, C17 SPH, 17C16 Cer, 18C17 Cer, 18C6-SM, 18C17-SM	1µM (S1P, SPH, Cer); 5µM SM

Instrumentation and method

Pump

• Type: Surveyor quaternary HPLC pump
• Mode: gradient elution
• Solvent(s): Solvent A: 1 mM Ammonium formate in Methanol with 0.2% formic acid; solvent B: 2 mM Ammonium formate in water with 0.2% formic acid
• Gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	0.5	80	20
4.5	0.5	90	10
7	0.5	90	10
28	0.5	99	1
29	0.5	80	20
33	0.5	80	20

Autosampler

• Type: Surveyor
• Injection volume: 20µl
• Wash solvent: n.d.

Column

• Type: BDS Hypersil C8 (Phenomenex)
• length: 150 x 3.2 mm i.d.
• Particle size: 3 µm
• Retention time: 4.92 min (S1P), 4.3 min (C17-S1P); 2.16 min (SPH), 2.44 min (C17-SPH); 14-18 min (Cer), 3.5-14.5 min (SM)

Mass spectrometer

• Type: Triple quadrupole (Thermo Finnigan)
• Ionization mode: ESI positive
• Ionization voltage: n.d.
• Source temperature: n.d.
• Collision gas: n.d.
• Collision gas pressure:
• MS/MS-conditions:

• • multiple reaction monitoring table of species observed frequently

Analyte	Precursor [m/z]	Precursor [m/z]	Collision energy [eV]
S1P	380.1	264.2	23eV
S1P 17:0	366.2	250	20eV
SPH	300.4	282.2	18eV
C17 SPH	286.1	268	17eV

Data analysis and quantification

Software

• n.d.

Calibration and quantification

• calibration type: standard addition without matrix for plasma and in the presence of total lipids extracted from HPAECs.
• species used for calibration

Species	Serum, tissue, cells
SPH, S1P	1-250pmol
Cers	2.5-400pmol
SMs	12.5-2000pmol

Method Validation

Accuracy

n.d.

Precision

n.d.

LOD

n.d.

Recovery

n.d.

Sample data

Mass spectra

Chromatogram

Reference

• Bielawski J, Szulc ZM, Hannun YA, Bielawska A Methods 2006, 39(2), 82-91.