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

LC Conditions

Column

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

Brand

Kinetex

Part No

00D-4759-AN

Phase Name

Polar C18

Elution Type

Gradient

Mobile Phase

A: 2mM Ammonium acetate (pH 6.9)

B: Methanol/Acetonitrile (50-50)

Gradient Profile

Step No.

Time(min)

9.5

9.51

Flow Rate

400.00 mL/min

Temperature

50°C

Detection

Mass Spectrometer (MS) @ (50°C)

Detection Info

Sample Notes

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

Sample Preparation Details

Description

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

Part No

CE0-7565

Pretreatment 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.