Extraction of Epinephrine, Norepinephrine and Dopamine from Human Plasma Using EVOLUTE® EXPRESS WCX Prior to LC-MS/MS Analysis

Introduction

Catecholamines are biomarkers used for the detection of diseases such as hypertension, pheochromocytoma and neuroblastoma. The main target analytes are dopamine, epinephrine and norepinephrine (see Figure 1. for details), which are traditionally analyzed using liquid chromatography with electrochemical detection.

Figure 1. Structures of catecholamines.

This application note describes a mixed-mode weak cation exchange solid phase extraction protocol using EVOLUTE^® EXPRESS WCX 96-well plates for the extraction of three catecholamines (epinephrine, norepinephrine and dopamine) from human plasma prior to LC-MS/MS detection, which allows low level quantification of the analytes. EVOLUTE EXPRESS solid phase extraction products combine powerful EVOLUTE sorbent chemistry with enhanced ‘EXPRESS’ column components. EVOLUTE EXPRESS products dramatically improve flow characteristics, and enhance sample preparation productivity. By truly eliminating the need for column conditioning and equilibration, samples can be prepared using a simple, fast load-wash-elute procedure.Analytes Epinephrine, Norepinephrine and Dopamine Internal Standards
D₆ Epinephrine, D₆ Norepinephrine, D₄ Dopamine (LGC, Teddington, UK). See Notes for further details on sample and standard preparation.Format:
EVOLUTE^® EXPRESS WCX 10 mg fixed well plate, part number 602-0010-PX01 Sample Pre-treatment
Mix plasma sample (250 μL) containing internal standard with 0.05% (v/v) formic acid (250 μL). Condition
Optional. Not required in load-wash-elute procedure.
If desired condition wells with methanol (0.5 mL). Equilibration
Optional. Not required in load-wash-elute procedure.
If desired equilibrate wells with 10mM ammonium acetate (0.5 mL). Sample Loading
Load 500 μL of pre-treated sample. Wash 1
Elute interferences with ammonium acetate (10 mM, 0.5 mL) Wash 2
Elute interferences with propan-2-ol (0.5 mL). Dry thoroughly (e.g. 50 psi, 5 mins, using Biotage^® PRESSURE+ 96). Elution
Elute analytes with 0.1% formic acid in water : propan-2-ol (85:15) (0.2mL), dry thoroughly as above. Note
To ensure high analyte recoveries and low RSDs, the SPE plate is dried thoroughly immediately before and after elution. Post Elution
Due to the low elution volume, no evaporation of the extract is required prior to analysis.

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