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Biotage Application Note
A new, efficient, effective and sensitive sample preparation application for the extraction of the catecholamines epinephrine, norepinephrine and dopamine from human plasma at significantly low levels using EVOLUTE WCX; a resin-based mixed-mode sorbent with an optimized combination of non-polar (hydrophobic) and weak cation exchange interactions.
Catecholamines are standard biomarkers for the detection of diseases such as hypertention, pheochromocytoma and neuroblastoma. Detection of these analytes in biological matrices (serum, plasma and urine) at sub-nanogram per millilitre levels is critical in the clinical diagnosis of these and other diseases.
This application note outlines the procedure using the EVOLUTE WCX 50 mg 3 mL column (part #612-0002-B) optimized for a 500 μL plasma sample volume, but other formats are available. Method parameters and dilution factors have been optimized to maximize recoveries and minimize ion suppression.
Sample pre-treatment: Dilute 500 μL of plasma 1:1 (v/v) with 50mM ammonium acetate, pH 7. Spike sample with internal standard.
Column conditioning: Condition column with methanol (1 mL).
Column equilibration: Rinse column with 50 mM ammonium acetate, pH 7 (1 mL).
Sample loading: Load pre-treated plasma sample (1 mL) at a flow-rate of 1–2 mL/min.
Interference elution 1: Remove polar and ionic interferences with methanol:water (10:90) (2 mL).
Interference elution 2: Remove non-polar and phospholipid interferences with isopropanol (2 mL).
Analyte elution: Elute catecholamines with methanol containing formic acid (95:5, v/v) (1 mL).
Post extraction: Evaporate to dryness and reconstitute sample in mobile phase (200 uL).
The extracted samples were run on an Agilent 1200 Liquid Handling System with a Restek Ultra PFP Propyl 3 μm analytical column (50 × 2.1 mm i.d.) at ambient temperature. The mobile phase was isocratic, 0.1% formic acid aq/methanol (98/2, v/v) at a flow-rate of 0.200 mL/min for 5 minutes. The detector was an Applied Biosystems/MDS Sciex 4000 Q-Trap triple quadrupole mass spectrometer equipped with a Turbo Ionspray interface for mass analysis. Negative ions were acquired in the multiple reaction monitoring mode (MRM); norepinephrine 170.2>152.2, epinephrine 184.1>166.1 and dopamine 154.3>137.3.
Figure 1: Extracted Ion Chromatogram for sub-nanogram levels of epinephrine (100 pg/mL) and dopamine (200 pg/mL) and norepinephrine (1000 pg/mL) extracted from spiked plasma with EVOLUTE WCX. The peaks denoted by * are plasma artefact peaks observed at the LLOD.
Sample recoveries of over 85% with RSDs below 10% were achieved at pg/mL levels for epinephrine and dopamine and 1 ng/mL for norepinephrine. Figure 1 shows the extracted ion chromatogram.
This method demonstrates a robust and effective extraction of a range of catecholamines at significantly low levels from the challenging biological matrix of human plasma. Some artefact peaks from the plasma were observed in the total ion chromatogram but these had no effect on the ions of interest, resulting in a clean and interference free chromatogram.
1. V.E. Vandell, Extraction of Sub-Nanogram Levels of Catecholamines from Human Plasma Using EVOLUTE WCX, Application Note AN 739 (available from www.biotage.com/applications).
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