Bile acids - LC-MS/MS - Scherer et al.

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10µL of IS-mixture (2.4µmol/L D4-CA, 2.6µmol/L D4-DCA, 4µmol/L D4-CDCA, 0.6µmol/L D4-LCA, 2.6µmol/L D4-UDCA, 4.6µmol/L D4-GCA, 14µmol/L D4-GCDCA).  
10µL of IS-mixture (2.4µmol/L D4-CA, 2.6µmol/L D4-DCA, 4µmol/L D4-CDCA, 0.6µmol/L D4-LCA, 2.6µmol/L D4-UDCA, 4.6µmol/L D4-GCA, 14µmol/L D4-GCDCA).  
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! Material  
! Material  
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! Human plasma or serum  
! Human plasma or serum  
| 100µl  
| 100µl  
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| 2.4µmol/L D</sup>4<sub>-CA, 2.6µmol/L D</sup>4<sub>-DCA, 4µmol/L D</sup>4<sub>-CDCA, 0.6µmol/L D</sup>4<sub>-LCA, 2.6µmol/L D</sup>4<sub>-UDCA, 4.6µmol/L D</sup>4<sub>-GCA, 14µmol/L D</sup>4<sub>-GCDCA
+
| 2.4µmol/L D<sub>4</sub>-CA, 2.6µmol/L D<sub>4</sub>-DCA, 4µmol/L D<sub>4</sub>-CDCA, 0.6µmol/L D<sub>4</sub>-LCA, 2.6µmol/L D<sub>4</sub>-UDCA, 4.6µmol/L D<sub>4</sub>-GCA, 14µmol/L D<sub>4</sub>-GCDCA
| 10µl
| 10µl
|}
|}

Revision as of 09:33, 27 April 2010

Contents

Sample preparation

  • 10µL of a methanolic IS-mixture were added to plasma
  • 30µL of 1mol/L HCl were added to serum/plasma
  • protein precipitation was performed with 1mL acetonitrile, followed by 1min vortex-mixing
  • after 15min of centrifugation (14,000×g), the supernatant was evaporated to dryness under reduced pressure
  • samples were re-dissolved in 100µL methanol/water (1/1, v/v, containing 10mmol/L ammonium acetate and 0.1% ammonium hydroxide)
  • after centrifugation the supernatant was used for analysis

10µL of IS-mixture (2.4µmol/L D4-CA, 2.6µmol/L D4-DCA, 4µmol/L D4-CDCA, 0.6µmol/L D4-LCA, 2.6µmol/L D4-UDCA, 4.6µmol/L D4-GCA, 14µmol/L D4-GCDCA).

Material Material used Internal Standard(s) Internal Standard(s) added
Human plasma or serum 100µl 2.4µmol/L D4-CA, 2.6µmol/L D4-DCA, 4µmol/L D4-CDCA, 0.6µmol/L D4-LCA, 2.6µmol/L D4-UDCA, 4.6µmol/L D4-GCA, 14µmol/L D4-GCDCA 10µl

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] Precursor [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.

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