LC/MS Analysis of Plasma Samples on Ascentis® Express C18 Showing Baseline Improvements after Phospholipid Removal using HybridSPE®-Phospholipid
Materials
SPE tube or plate
CONDITIONS
sample preparation
SPE (Solid Phase Extraction)
sample/matrix
rat plasma
SPE well plate
HybridSPE-Precipitation 96-well Plate, 50 mg/well (575656-U)
sample addition
100 μL rat plasma followed by 300 μL 1% formic acid in acetonitrile
elution
apply vacuum
column
Ascentis Express C18, 5 cm x 2.1 mm I.D., 2.7 μm particles (53822-U)
mobile phase
[A] 10 mM ammonium acetate; [B] 10 mM ammonium acetate in acetonitrile
gradient
0 min - 95% (A); 10 min - 50% (A); 18 min - 50% (A); 18.1 min - 95% (A); 22 min - 95% (A)
flow rate
0.5 mL/min
column temp.
35 °C
detector
ABI 3200QT; ESI (+), MRM (184/103 m/z)
injection
5 μL
Description
Application
HybridSPE-Precipitation:
Precipitate proteins by first adding 100 μL rat plasma to the HybridSPE-PPT plate followed by 300 μL 1% formic acid in acetonitrile. Do not apply vacuum.
Mix by vortexing the HybridSPE-PPT plate briefly.
Apply vacuum using a 96-well vacuum manifold and collect the resulting eluate for analysis.
Standard Protein Precipitation:
Combine 100 μL rat plasma with 300 μL 1% formic acid in acetonitrile.
Mix by vortexing and centrifuge.
Isolate resulting supernatant for analysis.
Legal Information
Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany
HybridSPE is a registered trademark of Merck KGaA, Darmstadt, Germany