Sphingolipid profiling - LC-MS/MS - Scherer et al.
From LipidomicsWiki
(Difference between revisions)
| Line 44: | Line 44: | ||
*Type: binary and isocratic pump (Agilent 1200) | *Type: binary and isocratic pump (Agilent 1200) | ||
*Mode: gradient elution | *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 | + | *Solvent(s): Solvent A water containing 0.2% formic acid and 200 mM ammonium formate ; Solvent B acetonitrile containing 0.2% formic acid |
*column flow was directed only from 1.0 to 3.0min into the mass spectrometer; otherwise methanol with a flow rate of 250µL/min was delivered | *column flow was directed only from 1.0 to 3.0min into the mass spectrometer; otherwise methanol with a flow rate of 250µL/min was delivered | ||
*Gradient: | *Gradient: | ||
| - | {| border="1" style="text-align: center; | + | {| border="1" class="wikitable" style="text-align: center;" |
|- | |- | ||
! Time [min] | ! Time [min] | ||
| Line 94: | Line 94: | ||
*Type: CTC Pal | *Type: CTC Pal | ||
| - | *Injection volume: | + | *Injection volume: 2µl |
*Wash solvent 1: Isopropanol/MeOH (1/1; v/v), Wash solvent 2: MeOH<br> | *Wash solvent 1: Isopropanol/MeOH (1/1; v/v), Wash solvent 2: MeOH<br> | ||
| Line 107: | Line 107: | ||
*Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP) | *Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP) | ||
| - | *Ionization mode: ESI | + | *Ionization mode: ESI positive |
| - | *Ionization voltage: | + | *Ionization voltage: 5500V |
| - | *Source temperature: | + | *Source temperature: 400°C |
*Collision gas: Nitrogen | *Collision gas: Nitrogen | ||
*Collision gas pressure: medium | *Collision gas pressure: medium | ||
*MS/MS-conditions: | *MS/MS-conditions: | ||
| - | **precursor ion scan of m/z | + | **precursor ion scan of m/z 264 for HexCer, LacCer, Cer1P species screening |
| + | **neutral loss scan of m/z 98 for dhCer1P species screening | ||
**multiple reaction monitoring table of species observed frequently | **multiple reaction monitoring table of species observed frequently | ||
| - | {| border="1 | + | {| border="1" style="text-align: center;" class="wikitable" |
|- | |- | ||
! Analyte | ! Analyte | ||
| Line 123: | Line 124: | ||
! Collision energy [eV] | ! Collision energy [eV] | ||
|- | |- | ||
| - | ! | + | ! SPH d18:1 |
| - | | | + | | 300.3 |
| - | | | + | | 282.2 |
| - | | | + | | 17 |
|- | |- | ||
| - | ! | + | ! |
| - | | | + | | 300.3 |
| - | | | + | | 252.2 |
| - | | | + | | 25 |
|- | |- | ||
| - | ! | + | ! SPH d18:0 |
| - | | | + | | 302.3 |
| - | | | + | | 284.2 |
| - | | | + | | 21 |
|- | |- | ||
| - | ! | + | ! |
| - | | | + | | 302.3 |
| - | | | + | | 254.2 |
| - | | | + | | 29 |
|- | |- | ||
| - | ! | + | ! SPH t18:0 |
| - | | | + | | 318.3 |
| - | | | + | | 282.2 |
| - | | | + | | 23 |
|- | |- | ||
| - | ! | + | ! Dimethyl-SPH |
| 435.2 | | 435.2 | ||
| 153 | | 153 | ||
Revision as of 12:44, 27 April 2010
Contents |
Sample preparation
- sphingolipids 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
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
- column flow was directed only from 1.0 to 3.0min into the mass spectrometer; otherwise methanol with a flow rate of 250µL/min was delivered
- Gradient:
| Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
|---|---|---|---|
| 0 | 0.8 | 0 | 100 |
| 0.1 | 0.8 | 0 | 100 |
| 0.11 | 0.8 | 10 | 90 |
| 2.5 | 0.8 | 50 | 50 |
| 3.5 | 0.8 | 50 | 50 |
| 3.51 | 0.8 | 0 | 100 |
| 4.5 | 0.8 | 0 | 100 |
Autosampler
- Type: CTC Pal
- Injection volume: 2µl
- 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 positive
- Ionization voltage: 5500V
- Source temperature: 400°C
- Collision gas: Nitrogen
- Collision gas pressure: medium
- MS/MS-conditions:
- precursor ion scan of m/z 264 for HexCer, LacCer, Cer1P species screening
- neutral loss scan of m/z 98 for dhCer1P species screening
- multiple reaction monitoring table of species observed frequently
| Analyte | Precursor [m/z] | Product [m/z] | Collision energy [eV] |
|---|---|---|---|
| SPH d18:1 | 300.3 | 282.2 | 17 |
| 300.3 | 252.2 | 25 | |
| SPH d18:0 | 302.3 | 284.2 | 21 |
| 302.3 | 254.2 | 29 | |
| SPH t18:0 | 318.3 | 282.2 | 23 |
| Dimethyl-SPH | 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
