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Extraction of unconjugated Bile acids from Human Serum on Kinetex 2.6µm Polar C18 100x2.1 Column

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Sample Preparation Details

Part Number :CE0-7565

Description :Impact Protein Precipitation, 2mL Square Well Filter Plate, 2/Pk

Load :Load Sample

Evaporate: :Evaporate to dryness

Reconstitute :0 µL initial mobile phase

Notes :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.

Application Detail (App ID: 24195)

Column: Kinetex® 2.6 µm Polar C18 100 Å, LC Column 100 x 2.1 mm, Ea

Part No:00D-4759-AN

Dimensions:100 x 2.1 mm ID

Elution Type:Gradient

Elution A:2mM Ammonium acetate (pH 6.9)

Elution B:Methanol/Acetonitrile (50-50)

Gradient Profile:

Step No.	Time (min)	Pct A	Pct B
1	0	55	45
2	9	30	70
3	9.5	30	70
4	9.51	55	45
5	12	55	45

Flow Rate:400 mL/min

Col. Temp: 50 °C

Detection: Mass Spectrometer (MS) @ amu (50°C)

Detector Info: SCIEX 4500 QTRAP®

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

Analyte Details

Analyte Detail not available

UDCA

UDCA-D4

GCDCA

GCDCA-D4

CA-D4

GDCA

GDCA-D4

TDCA

TDCA-D4

CDCA

CDCA-D4

DCA

DCA-D4

LCA

LCA-D4