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Extraction of Epinephrine Norepinephrine andDopamine from Human Plasma Using EVOLUTE EXPRESS WCX Prior to LC MS MS Analysis

IP.com Disclosure Number: IPCOM000243953D
Publication Date: 2015-Oct-30
Document File: 5 page(s) / 217K

Publishing Venue

The IP.com Prior Art Database

Abstract

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

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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

Epinephrine

Sample Preparation Procedure

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.

Norepinephrine Dopamine

Figure 1. Structures of catecholamines.

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.

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.

© Biotage 2015


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Extraction of Epinephrine, Norepinephrine and Dopamine from Human Plasma Using EVOLUTE EXPRESS WCX | Page 2

MS Conditions

HPLC Conditions

Instrument

Shimadzu Nexera UHPLC system

Column

ACE Excel 1.7 C18 PFP 100 x 2.1 mm

Mobile Phase

A: Water containing 0.25mM ammonium acetate and formic acid

B: Methanol containing 0.25...