| 1UDCA |
|---|
| 2UDCA-D4 |
| 3GCDCA |
| 4GCDCA-D4 |
| 5CA |
| 6CA-D4 |
| 7GDCA |
| 8GDCA-D4 |
| 9TDCA |
| 10TDCA-D4 |
| 11CDCA |
| 12CDCA-D4 |
| 13DCA |
| 14DCA-D4 |
| 15LCA |
| 16LCA-D4 |
Compound Name Actigall |
CID 465690 |
Molecular Formula C24H40O4 |
Molecular Weight 392 |
No. Chiral Centers: 1 |
No. Hydrogen Bond Acceptors 4 |
No. Hydrogen Bond Donors 3 |
Column: | ||||||||||||||||||||||||
Brand Name: Kinetex | ||||||||||||||||||||||||
Part No: | ||||||||||||||||||||||||
Phase Name: Polar C18 | ||||||||||||||||||||||||
Elution Type: Gradient | ||||||||||||||||||||||||
Mobile Phase: A: 2mM Ammonium acetate (pH 6.9) B: Methanol/Acetonitrile (50-50) | ||||||||||||||||||||||||
Gradient Profile:
| ||||||||||||||||||||||||
Flow Rate: 400 mL/min | ||||||||||||||||||||||||
Temperature: 50°C | ||||||||||||||||||||||||
Detection: Mass Spectrometer (MS) (50°C) | ||||||||||||||||||||||||
Detector Info: SCIEX 4500 QTRAP® | ||||||||||||||||||||||||
Sample Note: Sample Prep Protocol
Dispense: 400 uL methanol into the wells of the Impact plate
Add: 100 uL of doubly stripped Serum sample (spiked with analytes at 200ng/mL) directly into the organic solvent in each well of the plate.
Vortex: 2 minutes at maximum possible speed.
Wait: Allow 5 minutes for completion of protein precipitation.
Vacuum: Place the Impact plate onto a suitable 96-well SPE manifold followed by positioning a 96-well collection plate inside, under the Impact plate. Vacuum at 5" of Hg until filtrate is collected completely.
Dilute & inject: Dispense 300 uL of mobile phase A (or water) into the collection plate, vortex for 30 secs at a high speed and inject on LC-MS-MS
Note: A doubly stripped serum sample was employed for extraction purposes to eliminate the potential bias due to presence of any endogenous bile acids, leading to erroneous analyte quantitation.
Table 1. % Absolute Recovery for Bile acids from Human Serum Extraction on an Impact Protein Precipitation Plate
Analyte % Recovery % CV (N=5)
UDCA 91% 1.1
GCDCA 90% 3.7
CA 88% 4.8
GDCA 90% 4.4
TDCA 94% 3.5
CDCA 90% 4.5
DCA 88 4.6
LCA 90% 6.9 |
Description: Impact Protein Precipitation, 2mL Square Well Filter Plate, 2/Pk |
Part Number: |
Pre Treatment Note: Sample Prep Protocol
Dispense: 400 uL methanol into the wells of the Impact plate
Add: 100 ?L of doubly stripped Serum sample (spiked with analytes at 200ng/mL) directly into the organic solvent in each well of the plate.
Vortex: 2 minutes at maximum possible speed.
Wait: Allow 5 minutes for completion of protein precipitation.
Vacuum: Place the Impact plate onto a suitable 96-well SPE manifold followed by positioning a 96-well collection plate inside, under the Impact plate. Vacuum at 5" of Hg until filtrate is collected completely.
Dilute & inject: Dispense 300 uL of mobile phase A (or water) into the collection plate, vortex for 30 secs at a high speed and inject on LC-MS-MS
Note: A doubly stripped serum sample was employed for extraction purposes to eliminate the potential bias due to presence of any endogenous bile acids, leading to erroneous analyte quantitation. |
Sample PreTreatment: Sample Prep Protocol
Dispense: 400 uL methanol into the wells of the Impact plate
Add: 100 ?L of doubly stripped Serum sample (spiked with analytes at 200ng/mL) directly into the organic solvent in each well of the plate.
Vortex: 2 minutes at maximum possible speed.
Wait: Allow 5 minutes for completion of protein precipitation.
Vacuum: Place the Impact plate onto a suitable 96-well SPE manifold followed by positioning a 96-well collection plate inside, under the Impact plate. Vacuum at 5" of Hg until filtrate is collected completely.
Dilute & inject: Dispense 300 uL of mobile phase A (or water) into the collection plate, vortex for 30 secs at a high speed and inject on LC-MS-MS
Note: A doubly stripped serum sample was employed for extraction purposes to eliminate the potential bias due to presence of any endogenous bile acids, leading to erroneous analyte quantitation. |
Evaporation: Evaporate to dryness |
