Sphingosine-1-phosphate, Lysophosphatidic acid - LC-MS/MS - Scherer et al.

From LipidomicsWiki

Revision as of 12:22, 27 April 2010 (view source) Gerhard Liebisch (Talk | contribs) ← Previous diff		Current revision (15:17, 27 April 2010) (view source) Gerhard Liebisch (Talk | contribs)
Line 147:		Line 147:
	*Analyst 1.4.2.		*Analyst 1.4.2.
		+	*isotope correction as described for [[Phosphatidylcholine / Sphingomyelin - ESI-MS/MS - Liebisch et al.|Phosphatidylcholine / Sphingomyelin - ESI-MS/MS - Liebisch et al.]]
	=== Calibration and quantification  ===		=== Calibration and quantification  ===

---

Current revision

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

• S1P and LPA were extracted with butanol
• internal standards were added prior lipid extraction
• supernatant was transferred to a glass vial and injected
  

Material	Material used	Internal Standard(s)	Internal Standard(s) added
Human plasma	75µl	13C2D2-S1P, LPA 17:0	20 ng

Instrumentation and method

Pump

• Type: binary and isocratic pump (Agilent 1200)
• Mode: gradient elution
• Solvent(s): Solvent A 90mM ammonium formate/formic acid (100/0.2 v/v); Solvent B acetonitril/formic acid (100/0.2 v/v)
• Gradient:

Time [min]	Flow [ml/min]	% Solvent A	% Solvent B
0	0.8	5	95
0.5	0.8	5	95
1.5	0.8	50	50
1.7	0.8	50	50
1.9	0.8	5	95
2.2	0.8	5	95

Autosampler

• Type: CTC Pal
• Injection volume: 10µl
• Wash solvent 1: Isopropanol/MeOH (1/1; v/v), Wash solvent 2: MeOH
  

Column

• Type: Interchim HILIC column (Interchim, Montlucan, France)
• length: 50 x 2 mm i.d., pre-column filter
• Particle size: 2.2 µm

Mass spectrometer

• Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
• Ionization mode: ESI negative
• Ionization voltage: -4500V
• Source temperature: 300°C
• Collision gas: Nitrogen
• Collision gas pressure: medium
• MS/MS-conditions:
  • precursor ion scan of m/z 153 for LPA species screening
  • multiple reaction monitoring table of species observed frequently

Analyte	Precursor [m/z]	Product [m/z]	Collision energy [eV]
S1P	378.2	79	58V
SA1P	380.2	79	58V
13C2D2-S1P	382.2	79	58V
LPA 16:0	409.2	153	30V
LPA 18:0	437.2	153	30V
LPA 18:1	435.2	153	30V
LPA 18:2	433.2	153	30V
LPA 20:4	457.2	153	30V
LPA 17:0	423.2	153	30V

Data analysis and quantification

Software

• Analyst 1.4.2.
• isotope correction as described for Phosphatidylcholine / Sphingomyelin - ESI-MS/MS - Liebisch et al.

Calibration and quantification

• calibration type: matrix calibration - addition of naturally occurring species
• species used for calibration

Species	Human plasma
S1P	0.352-3.518µmol/L
SA1P	0.175-1.75µmol/L
LPA 16:0	0.162-1.62µmol/L
LPA 18:0	0.0304-0.304µmol/L
LPA 18:1	0.0917-0.917µmol/L
LPA 18:2	0.308-3.08µmol/L
LPA 20:4	0.291-2.91µmol/L

Method Validation

Accuracy

90 - 106 %

Precision

CV: 3 % - 10 %

LOD

6 nmol/L for S1P, SA1P and <2nmol/l

Recovery

85% (S1P, SA1P); 80 % LPA 

Sample data

Mass spectra

Chromatogram 

Reference

Scherer et al. Clin Chem. 2009 Jun;55(6):1218-22.