Lee Williams, Matthew Cleeve, Scott Merriman, Helen Lodder, Rhys Jones, Steve Jordan, Steve Plant, Richard Calverley and Joanna
Caulfield, Biotage AB, Uppsala, Sweden.
Introduction
This application note from Biotage describes the effective removal of two major classes of matrix components, proteins and
phospholipids, using EVOLUTE CX. Gel electrophoresis and ion suppression data are shown, comparing EVOLUTE CX with two other
commercially available mixed-mode resin based products.
EVOLUTE CX Sample Preparation
EVOLUTE CX configuration: EVOLUTE CX 25 mg fixed-well plate (part number 601-0025-P01)
Sample pre-treatment: Plasma sample (100 μL) was diluted with 50 mM ammonium acetate buffer at pH 6 (1:3, v/v)
Column conditioning: Methanol (1 mL)
Column equilibration: 50 mM ammonium acetate buffer at pH 6 (1 mL)
Sample loading: Pre-treated plasma sample (400 μL)
Interference elution 1: 50 mM ammonium acetate buffer at pH 6 (1 mL)
Interference elution 2: Methanol (1 mL)
Analyte elution: 5% (v/v) NH4OH in methanol (1 mL) Published methods were used for the competitor products.
Gel electrophoresis
Full details are available from Biotage on request.1
Phospholipid removal
Sample extracts were reconstituted in 1 mL of 70:30 (v/v) H2O/MeOH prior to analysis.
HPLC Conditons
Instrument: Waters 2795 Liquid Handling System (Waters Assoc., Milford, Massachusetts, USA).
Column: Luna Phenyl-Hexyl 5 μm analytical column (50 × 2.0 mm i.d.) equipped with a Security guard column (both Phenomenex, Cheshire
UK).
Mobile phase: 0.1% formic acid aq. and 0.1% formic acid in MeCN at a flow-rate of 0.3 mL/min.
Gradient: The gradient conditions were set to 60%, 0.1% (v/v) formic acid aq. and 40% MeCN increasing to 100% MeCN over 6 minutes.
The high organic mobile phase was held for 7 minutes and initial starting conditions resumed at 13.1 minutes.
Injection volume: 5 μL
Temperature: Ambient
Results
Figure 1
|
Gel electrophoresis
Figure 1 shows the gel electrophoresis profile for the three polymer-based SPE products compared to raw serum and a protein
benchmark molecular weight ladder. The results show that all three products provide greater than 99% serum protein removal.
Figure 2
|
Phospholipid removal
Figure 2 shows the amount of phospholipids present in the final extracts of the various SPE sorbents compared to a protein
precipitated extract. The bottom scan function corresponds to Lyso-phospholipid elution, while the more retentive phospholipids
are shown in the top scan function.
Conclusions
EVOLUTE CX Mixed-mode Cation Exchange SPE provides superior removal of the two major matrix components from plasma, in particular
the most challenging of them all: phospholipids.
References
1. Poster presented at BMSS 2008: Comparison of Plasma Extract Cleanliness using Resin-based Mixed-mode Cation Exchange SPE
Sorbents, Lee Williams et al.
EVOLUTE is a registered trademark of Biotage.
Biotage AB
Kungsgatan 76, SE-753 18 Uppsala, Sweden
tel. +46 18 56 57 10 fax +46 18 56 57 05
E-mail: order@eu.biotage.com
Website:
http://www.biotage.com/
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