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Plasma Free Metanephrines Quantitation
with Automated Online Sample Preparation
and Liquid Chromatography—Tandem
Mass Spectrometry
Xiang He and Marta Kozak, Thermo Fisher Scientific, San Jose, CA
Application Note 563
Key Words
TSQ Vantage, Clinical Research, TurboFlow Technology, Metanephrine, MN,
Normetanephrine, NMN, Pmets, Pheochromocytoma
Goal
To develop an automated method to quantitate plasma free metanephrines
reducing method time while maintaining analytical performance compared
to the original offline SPE method.
Introduction
Plasma free metanephrine (MN) and normetanephrine
(NMN), collectively known as Pmets, are preferred
biomarkers for pheochromocytoma for clinical research.
Liquid chromatography-tandem mass spectrometry
(LC-MS/MS) has become widely used to measure Pmets
because of its high analytical specificity.
Recently, we reported an LC-MS/MS method for
measuring Pmets using ion-pairing solid phase extraction
(IP-SPE) and porous graphitic carbon (PGC) column
chromatography
1, 2
. Although the method is fast and
analytically sensitive, it can be further improved by
automating the offline sample preparation with online
sample preparation technology, which is more time- and
cost-effective.
Thermo Scientific TurboFlow technology is an automated
online sample preparation technology that has been
coupled to LC-MS/MS for the quantitative analysis of a
variety of biological samples.
To date, its use has been reported in clinical research,
pharmaceutical analysis, bioanalysis, environmental
testing, food safety, and forensic toxicology.
Methods
Sample Preparation
The 0.5-mL samples of human plasma and of charcoal
stripped serum (CSS) were spiked with internal standards
(IS) and then mixed with 0.25 mL of 10% tricholoacetic
acid (w/v) in water. The mixtures were vortexed and
stored at –30 °C for 30 minutes. Then, the mixtures were
centrifuged at 16,000 g for 10 minutes, and 100 µL of the
supernatants were injected for LC-MS/MS analysis.
LC-MS/MS Conditions
LC-MS/MS analysis was performed on a Thermo Scientific
TSQ Vantage triple stage quadrupole mass spectrometer
coupled with a Thermo Scientific Transcend TLX-1 system.
The TurboFlow
™
method with automated online sample
preparation was performed with a TurboFlow Cyclone
MCX-2 column. Perfluoroheptanoic acid (PFHA) was used
as the ion-pair during the sample preparation.
Figure 1. TurboFlow and
LC method