Bile acid quantitation - LC-MS/MS

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Contents

Sample preparation

  • 100µl of sample are spiked with 100µl internal standard solution and 800µl of acetonitrile are added. The samples are centrifuged and the supernatant is evaporated. Samples are taken up in 100µl of starting solvent.
  • sample sovlent: Solvent A / Solvent B  40:60 
Material Material used Internal Standard(s) Internal Standard(s) added
Serum  100µl

d4-Deoxycholic Acid, d-4 Lithocholic Acid,

d-4 Glycocholic Aicd, d4-Glycochenodeoxycholic Acid

10pmol each

Instrumentation and method

Pump

  • Type: Binary Agilent 1100
  • Mode: Gradient
  • Solvent A: Water
  • Solvent B: Methanol (5mM amonia acetate, 0.12% formic acid)
  • Gradient:
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.3 40  60
5.0 0.3 25  75 
12.0 0.3 15  85 
12.5 0.3 95 
16.0 0.3 95 

Column

  • Type: Zorbax Extend C18, 50x2.1 mm, 3.5µm
  • Temperature: 20°C

Autosampler

  • Type: Agilent 1100
  • Injection volume: 20ul
  • Wash solvent: Methanol

Mass spectrometer

  • Type: 4000QTrap
  • Ionization mode: negative ESI
  • Ionization voltage: 4000V
  • Source temperature: 590°C
  • Gas 1: 50psi
  • Gas 2: 70psi
  • Collision gas: Nitrogen
  • MS/MS-conditions:
    • multiple reaction monitoring table
Analyte Precursor [m/z] Fragment [m/z] Collision energy [eV]
Cholic Acid  407  407  40 
Deoxycholic Acid  391  391  40 
Chenodeoxycholic Acid  391  391  40 
Ursodeoxycholic Acid  391  391  40 
Litocholic Acid  375  375  40 
Taurocholic Acid  514  80  115
Taurodeoxycholic Acid  498  80  115
Taurochenodeoxycholic Acid  498  80  115 
Tauroursodeoxycholic Acid  498  80  115 
Taurolitocholic Acid  482  80  115 
Glycocholic Acid  464  74  70 
Glycodeoxycholic Acid  448  74  70 
Glycochenodeoxycholic Acid  448  74  7
Glycoursodeoxycholic Acid  448  74  70 
Glycolitocholic Acid  432  74  95 

Data analysis and quantitation

Data handling

  • Data processing is done automatically by InteliQuan 

Calibration and quantitation

  • calibration type: Standard curve
  • Calibration range: 5 - 1000 nM 

Method validation

Accuracy

Mean accuracy of 5 replicates is between 80% and 120% except for glycoconjugates at the LLOQ

Precision

Method precision for 5 replicates is better than 15%

Limit of quantitation

100fmol on coumn

Reference

Lipid_mass_spectrometry Bile_acids_and_derivatives_(ST04)

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