Sphingolipid profiling - LC-MS/MS - Scherer et al.

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Revision as of 12:29, 27 April 2010

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
Cell homogenate	100µg	SPH d17:1	20 ng
		SPC d17:1	2 ng
		GluCer 12:0	20 ng
		LacCer 12:0	20 ng
		Cer1P 12:0	20 ng

Instrumentation and method

Gradient elution was performed with 100% B for 0.1 min, a step to 90% B until 0.11 min, a linear increase to 50% B until 2.5 min, 50% B until 3.5 min and re-equilibration from 3.51 to 4.5 min with 100% B. The flow rate was set to 800 µL/min.

Pump

Type: binary and isocratic pump (Agilent 1200)
Mode: gradient elution
Solvent(s): Solvent A water containing 0.2% formic acid and 200 mM ammonium formate ; Solvent B acetonitrile containing 0.2% formic acid
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., 0.5 µm pre-column filter
Particle size: 1.8 µm
Column temperature: 50°C

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.

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.

@@ Line 39: / Line 39: @@
 == Instrumentation and method  ==
+Gradient elution was performed with 100% B for 0.1 min, a step to 90% B until 0.11
+min, a linear increase to 50% B until 2.5 min, 50% B until 3.5 min and re-equilibration
+from 3.51 to 4.5 min with 100% B. The flow rate was set to 800 µL/min.
 === Pump  ===
@@ Line 44: / Line 49: @@
 *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)
+*Solvent(s): Solvent A water containing 0.2% formic acid and 200 mM ammonium formate ; Solvent B acetonitrile containing 0.2% formic acid
 *Gradient:
-{| border="1" class="wikitable" style="text-align: center;"
+{| border="1" style="text-align: center;" class="wikitable"
 |-
 ! Time [min]
@@ Line 94: / Line 99: @@
 *Type: Interchim HILIC column (Interchim, Montlucan, France)
-*length: 50 x 2 mm i.d., pre-column filter
+*length: 50 x 2 mm i.d., 0.5 µm pre-column filter
-*Particle size: 2.2 µm
+*Particle size: 1.8 µm
+*Column temperature: 50°C
 === Mass spectrometer  ===
@@ Line 109: / Line 115: @@
 **multiple reaction monitoring table of species observed frequently
-{| border="1" class="wikitable" style="text-align: center;"
+{| border="1" style="text-align: center;" class="wikitable"
 |-
 ! Analyte