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

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== Sample preparation ==
== Sample preparation ==
* S1P and related compounds were extracted with methanol precipitation
* S1P and related compounds were extracted with methanol precipitation
* internal standards were added prior lipid extraction
* internal standards were added prior lipid extraction
-
* supernetants were transferred to glass vials prior to injection into the ESI LC-MS/MS system
+
* supernatant was transferred to glass vials prior to injection into the ESI LC-MS/MS system
{| class="wikitable" style="text-align:center"
{| class="wikitable" style="text-align:center"
! Material !! Material used  !! Internal Standard(s) !! Internal Standard(s) added
! Material !! Material used  !! Internal Standard(s) !! Internal Standard(s) added
-
|-
 
-
!
 
-
|
 
|-
|-
! Human plasma
! Human plasma
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|-
|-
! 0  
! 0  
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| 0.3 || 57.5 ||42.5
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| 0.3 || 100 ||0
|-
|-
! 0.6  
! 0.6  
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=== Autosampler ===
=== Autosampler ===
* Type: CTC Pal
* Type: CTC Pal
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* Injection volume: 20µl
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* Injection volume: 5µl
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* Wash solvent: methanol/chloroform = 1/1
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* Wash solvent: n.d.
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 +
=== Column ===
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* Type: Luna RP C 18 with a C18 guard column (Phenomenex, Aschaffenburg, Germany)
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* length: 150 x 2 mm i.d., guard column 4 x 2 mm i.d. 
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* Particle size: 5 µm
=== Mass spectrometer ===
=== Mass spectrometer ===
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* Type: Triple quadrupole (Micromass, Quattro Ultima)
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* Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
* Ionization mode: ESI positive
* Ionization mode: ESI positive
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* Ionization voltage:
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* Ionization voltage: 5400V
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* Source temperature:
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* Source temperature: 400°C
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* Collision gas: Argon
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* Collision gas: Nitrogen
* Collision gas pressure:  
* Collision gas pressure:  
* MS/MS-conditions:
* MS/MS-conditions:
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! Analyte !! Precursor [m/z] !! Precursor [m/z] !! Collision energy [eV]  
! Analyte !! Precursor [m/z] !! Precursor [m/z] !! Collision energy [eV]  
|-
|-
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! Cer 14:0 - H2O
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! S1P
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| 492.5|| 264.2 ||x
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| 380.1|| 264.2 ||23V
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|-
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! S1P 17:0
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| 366.2|| 250.1 ||23V
|-
|-
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! Cer 14:0
 
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| 510.5|| 264.2 ||x
 
|-
|-
|}
|}
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== Sample Preparation ==
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== Data analysis and quantification ==
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S1P and related compounds were extracted with methanol
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precipitation. Therefore, the prepared standard mixture were
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centrifuged at 10,000 x g for 5 min, and supernatants were
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transferred to glass vials prior to injection into the ESI-LC-MS/MS
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system.
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== Analytical Method ==
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=== Software ===  
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=== HPLC-MS-MS ===
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* Analyst 1.4.2.
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==== HPLC ====
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HPLC: Agilent 1100 Series binary pump (G1312A); Agilent degasser (G13791A); HTC Pal autosampler (Chromtech)
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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.)
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=== Calibration and quantification ===
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* calibration type: matrix calibration - addition of naturally occurring species
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eluent A: water/formic acid (100/0.1 v/v);
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* species used for calibration
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eluent B: acetonitril/ tetrahydrofuran/formic acid (50/50/0.1 v/v)
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{| class="wikitable" style="text-align:center"
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! Species!! Human plasma
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linear gradient: A/B = 57.5/42.5 (0 - 0.6 min)
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|-
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A/B = 0/100 (5 -10 min)
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! S1P
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A/B = 57.5/42.5 (10.5 - 14 min)
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| 50-1000ng/mL
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|-
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flow rate: 0.3 ml/min;
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|}
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injection volume: 5 μl
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==== MS 4000 Q-Trap (Applied Biosystems)====
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triple quadrupole; 
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Turbo V source ion spray;
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positive ion mode;
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electrospray voltage: 5400 V;
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source temperature: 400 °C;
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auxiliary gas: 30 psi;
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nebulizer gas: 30 psi;
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curtain gas: 10 psi;
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collision gas: 4 psi;
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dwell time: 100 ms
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All quadrupoles were working at unit resolution
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linear ion trap.
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Quantitation was performed with Analyst Software V1.4 (Applied
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-
Biosystems).
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== Method Validation ==
== Method Validation ==
=== Accuracy ===
=== Accuracy ===
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100.6 ± 6.7 (S1P);
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100.6 ± 6.7 (S1P)
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100.8 ± 4.8 (SPH);
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100.6 ± 3.6 (SAPH);
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99.7 ± 3.1 (SA1P)
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=== Precision ===
=== Precision ===
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interday and intraday precision
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intraday 96.2 - 104.4 % and interday precision 96.3 - 104.3 %
=== LOD ===
=== LOD ===
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n.d.
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<10.2 ng/mL for S1P
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=== LOQ ===
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'''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
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=== Recovery ===
=== Recovery ===
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84.3 ± 7.3 (S1P); 87,9 ± 8.2 (SPH); 85.7 ± 7.7 (SAPH);
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84.3 ± 7.3 (S1P)
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77.5 ± 10.5 (SA1P); 82.4 ± 11.9 (C17 S1P); 77.5 ± 10.5 (C17 SPH)
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 +
== Sample data ==
 +
 
 +
=== Mass spectra ===
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== Internal Standard ==
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=== Chromatogram ===
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C17 S1P for quantitation of S1P and SA1P;
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C17 SPH for quantitation of SPH and SAPH
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== Reference ==
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== Reference ==
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* H. Schmidt, R. Schmidt, G. Geisslinger ''Prostaglandins & other Lipid Mediators'' '''2006''', ''81'', 162-170.
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Pharmazentrum Frankfurt/ZAFES; Institut für Klinische Pharmakologie; Klinikum der Johann Wolfgang Goethe-Universität; Theodor-Stern-Kai 7; 60590 Frankfurt am Main; Germany.
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*[http://www.ncbi.nlm.nih.gov/pubmed/17085324?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum H. Schmidt, R. Schmidt, G. Geisslinger ''Prostaglandins &amp; other Lipid Mediators'' '''2006''', ''81'', 162-170.]
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Tel: +49-69-6301-7618; Fax: +49-69-6301-7636; e-mail: Helmut.Schmidt@em.uni-frankfurt.de (H. Schmidt)
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[[Category:LC-MS/MS]] [[Category:Triple_quadrupole]] [[Category:Sphingolipids_(SP)]] [[Category:Sphingoid_bases_(SP01)]] [[Category:ESI]] [[Category:HPLC]]

Current revision

Contents

Sample preparation

  • S1P and related compounds were extracted with methanol precipitation
  • internal standards were added prior lipid extraction
  • supernatant was 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 0.3 100 0
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.

Column

  • Type: Luna RP C 18 with a C18 guard column (Phenomenex, Aschaffenburg, Germany)
  • length: 150 x 2 mm i.d., guard column 4 x 2 mm i.d.
  • Particle size: 5 µm

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
S1P 17:0 366.2 250.1 23V

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 50-1000ng/mL

Method Validation

Accuracy

100.6 ± 6.7 (S1P)

Precision

intraday 96.2 - 104.4 % and interday precision 96.3 - 104.3 %

LOD

<10.2 ng/mL for S1P

Recovery

84.3 ± 7.3 (S1P)

Sample data

Mass spectra

Chromatogram

Reference

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