Sphingosine-1-phosphate - LC-MS/MS - Schmidt et al.
From LipidomicsWiki
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- | == | + | == Data analysis and quantification == |
- | + | ||
- | + | === Data handling === | |
- | + | * combine spectra above half peak height | |
- | + | * smooth combined spectrum (if necessary) | |
- | + | * centroid combined spectrum | |
+ | * pick peak intensities | ||
+ | |||
+ | === Isotope correction === | ||
+ | * correction of istopic overlap according to principles described for [[Phosphatidylcholine / Sphingomyelin determination - Liebisch et al.]] | ||
+ | |||
+ | === Calibration and quantification === | ||
+ | * calibration type: matrix calibration - addition of naturally occurring species | ||
+ | * both [M+H]+ and [M+H-H2O]+ are used | ||
+ | * species used for calibration | ||
+ | {| class="wikitable" style="text-align:center" | ||
+ | ! Species!! Cultured cells !! Human plasma | ||
+ | |- | ||
+ | ! Cer 16:0 | ||
+ | | 0-35pmol || 0-50pmol | ||
+ | |- | ||
+ | ! Cer 18:0 | ||
+ | | 0-35pmol || 0-50pmol | ||
+ | |- | ||
+ | ! Cer 20:0 | ||
+ | | 0-35pmol || 0-50pmol | ||
+ | |- | ||
+ | ! Cer 24:1 | ||
+ | | 0-35pmol || 0-50pmol | ||
+ | |- | ||
+ | ! Cer 24:0 | ||
+ | | 0-35pmol || 0-50pmol | ||
+ | |} | ||
== Analytical Method == | == Analytical Method == |
Revision as of 12:47, 21 July 2008
Contents |
Sample preparation
- S1P and related compounds were extracted with methanol precipitation
- internal standards were added prior lipid extraction
- supernetants were transferred to glass vials prior to injection into the ESI LC-MS/MS system
Material | Material used | Internal Standard(s) | Internal Standard(s) added |
---|---|---|---|
Human plasma | 50µl | S1P 17:0 | 7.5 ng |
Instrumentation and method
Pump
- Type: binary high pressure gradient (Agilent 1100)
- Mode: gradient elution
- Solvent(s): Solvent A water/formic acid (100/0.1 v/v); Solvent B acetonitril/ tetrahydrofuran/formic acid (50/50/0.1 v/v)
- Gradient:
Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
---|---|---|---|
0 | 0.3 | 57.5 | 42.5 |
0.6 | 0.3 | 57.5 | 42.5 |
5 | 0.3 | 0 | 100 |
10 | 0.3 | 0 | 100 |
10.5 | 0.3 | 57.5 | 42.5 |
14 | 0.3 | 57.5 | 42.5 |
Autosampler
- Type: CTC Pal
- Injection volume: 5µl
- Wash solvent: n.d.
Mass spectrometer
- Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
- Ionization mode: ESI positive
- Ionization voltage: 5400V
- Source temperature: 400°C
- Collision gas: Nitrogen
- Collision gas pressure:
- MS/MS-conditions:
- precursor ion scan of m/z 264.2
- multiple reaction monitoring table of species observed frequently
Analyte | Precursor [m/z] | Precursor [m/z] | Collision energy [eV] |
---|---|---|---|
S1P | 380.1 | 264.2 | 23V |
Data analysis and quantification
Data handling
- combine spectra above half peak height
- smooth combined spectrum (if necessary)
- centroid combined spectrum
- pick peak intensities
Isotope correction
- correction of istopic overlap according to principles described for Phosphatidylcholine / Sphingomyelin determination - Liebisch et al.
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- both [M+H]+ and [M+H-H2O]+ are used
- species used for calibration
Species | Cultured cells | Human plasma |
---|---|---|
Cer 16:0 | 0-35pmol | 0-50pmol |
Cer 18:0 | 0-35pmol | 0-50pmol |
Cer 20:0 | 0-35pmol | 0-50pmol |
Cer 24:1 | 0-35pmol | 0-50pmol |
Cer 24:0 | 0-35pmol | 0-50pmol |
Analytical Method
HPLC-MS-MS
HPLC
HPLC: Agilent 1100 Series binary pump (G1312A); Agilent degasser (G13791A); HTC Pal autosampler (Chromtech)
columns: Luna RP (150 mm L x 2 mm i.d., 5 µm particle size, 100 Å pore size); C18 guard column (4 mm Lx 2 mm i.d.)
eluent A: water/formic acid (100/0.1 v/v); eluent B: acetonitril/ tetrahydrofuran/formic acid (50/50/0.1 v/v)
linear gradient: A/B = 57.5/42.5 (0 - 0.6 min) A/B = 0/100 (5 -10 min) A/B = 57.5/42.5 (10.5 - 14 min)
flow rate: 0.3 ml/min; injection volume: 5 μl
MS 4000 Q-Trap (Applied Biosystems)
triple quadrupole; Turbo V source ion spray; positive ion mode;
electrospray voltage: 5400 V; source temperature: 400 °C; auxiliary gas: 30 psi; nebulizer gas: 30 psi; curtain gas: 10 psi; collision gas: 4 psi; dwell time: 100 ms
All quadrupoles were working at unit resolution linear ion trap. Quantitation was performed with Analyst Software V1.4 (Applied Biosystems).
Method Validation
Accuracy
100.6 ± 6.7 (S1P); 100.8 ± 4.8 (SPH); 100.6 ± 3.6 (SAPH); 99.7 ± 3.1 (SA1P)
Precision
interday and intraday precision
LOD
n.d.
LOQ
LLOQ: <10.2 ng/mL for S1P; <4.6 ng/mL for SPH; <1.9 ng/mL for SAPH; 0.57 ng/mL for SA1P
Recovery
84.3 ± 7.3 (S1P); 87,9 ± 8.2 (SPH); 85.7 ± 7.7 (SAPH); 77.5 ± 10.5 (SA1P); 82.4 ± 11.9 (C17 S1P); 77.5 ± 10.5 (C17 SPH)
Internal Standard
C17 S1P for quantitation of S1P and SA1P; C17 SPH for quantitation of SPH and SAPH
Reference
- H. Schmidt, R. Schmidt, G. Geisslinger Prostaglandins & other Lipid Mediators 2006, 81, 162-170.
Pharmazentrum Frankfurt/ZAFES; Institut für Klinische Pharmakologie; Klinikum der Johann Wolfgang Goethe-Universität; Theodor-Stern-Kai 7; 60590 Frankfurt am Main; Germany.
Tel: +49-69-6301-7618; Fax: +49-69-6301-7636; e-mail: Helmut.Schmidt@em.uni-frankfurt.de (H. Schmidt)