Bile acid quantitation - LC-MS/MS
From LipidomicsWiki
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 | 5 | 95 |
16.0 | 0.3 | 5 | 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 | 70 |
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