Extraction of Epinephrine Norepinephrine andDopamine from Human Plasma Using EVOLUTE EXPRESS WCX Prior to LC MS MS Analysis
Publication Date: 2015-Oct-30
The IP.com Prior Art Database
This application note describes a mixed mode weakcation exchange solid phase extraction protocol usingEVOLUTE EXPRESS WCX 96 well plates for the extractionof three catecholamines epinephrine norepinephrine anddopamine from human plasma prior to LC MS MS detection which allows low level quantification of the analytes
Page 01 of 5
Application Note AN844
Extraction of Epinephrine, Norepinephrine and Dopamine from Human Plasma Using EVOLUTE EXPRESS WCX | Page 1
Extraction of Epinephrine, Norepinephrine and Dopamine from Human Plasma Using EVOLUTE®
EXPRESS WCX Prior to LC-MS/MS Analysis
Sample Preparation Procedure
EVOLUTE EXPRESS WCX 10 mg fixed well plate, part number 602-0010-PX01
Mix plasma sample (250 μL) containing internal standard with
0.05% (v/v) formic acid (250 μL).
Optional. Not required in load-wash-elute procedure. If desired condition wells with methanol (0.5 mL).
Optional. Not required in load-wash-elute procedure.
If desired equilibrate wells with 10mM ammonium acetate
Load 500 µL of pre-treated sample.
Elute interferences with ammonium acetate (10 mM, 0.5 mL)
Elute interferences with propan-2-ol (0.5 mL). Dry thoroughly (e.g. 50 psi, 5 mins, using Biotage PRESSURE+ 96).
Elute analytes with 0.1% formic acid in water : propan-2-ol (85:15) (0.2mL), dry thoroughly as above.
To ensure high analyte recoveries and low RSDs, the SPE plate is dried thoroughly immediately before and after elution.
Due to the low elution volume, no evaporation of the extract is required prior to analysis.
Figure 1. Structures of catecholamines.
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.
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.
Epinephrine, Norepinephrine and Dopamine
D6 Epinephrine, D6 Norepinephrine, D4 Dopamine ( LGC, Teddington, UK).
See Notes for further details on sample and standard preparation.
© Biotage 2015
Page 02 of 5
Extraction of Epinephrine, Norepinephrine and Dopamine from Human Plasma Using EVOLUTE EXPRESS WCX | Page 2
Shimadzu Nexera UHPLC system
ACE Excel 1.7 C18 PFP 100 x 2.1 mm
A: Water containing 0.25mM ammonium acetate and formic acid
B: Methanol containing 0.25...