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Quantitation of Estrone and Estradiol
with Automated Online Sample Preparation
and LC-MS/MS
Xiang He and Marta Kozak, Thermo Fisher Scientific, San Jose, CA
Introduction
Estrone (E1) and estradiol (E2) are two major biologically
active estrogens. Quantitative measurements of these two
estrogens are important in clinical research.
Quantitation of serum estrogens has been performed
with immunoassay and gas chromatography-mass
spectrometry (GC-MS). Liquid chromatography-tandem
mass spectrometry (LC-MS/MS) is preferred over
immunoassay and other analytical techniques because
it is more analytically specific. Recently, we developed
a simple, fast and analytically sensitive method for
measuring underivatized E1 and E2 in serum or plasma
by LC-MS/MS using atmospheric pressure chemical
ionization (APCI).
1
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 the fields of clinical research, pharmaceutical
analysis, bioanalysis, environmental testing, food safety,
and forensic toxicology.
Goal
To develop a fast and analytically sensitive LC-MS/MS
method with automated online sample preparation for
simultaneous quantitation of underivatized E1 and E2 in
serum using TurboFlow
TM
technology.
Application
Note: 558
Key Words
• TSQ Vantage
• Transcend TLX
System
• Accucore RP-MS
Column
• Clinical Research
• TurboFlow
Technology
Methods
Sample Preparation
Briefly, 0.5 mL of sample was mixed with 0.5 mL of
working internal standard (E2-d5, IS) solution in methanol.
The mixture was vortexed, kept at -30 °C for 30 min and
then centrifuged at 16,000 g for 3 min at room temperature.
This process was repeated once for complete protein
precipitation. The supernatant (300 µL) was directly
injected for TurboFlow 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
equipped with Accela 1250 pumps. The online sample
preparation was performed with TurboFlow Cyclone-P
polymer-based columns.
Analytical high-performance
liquid chromatography (HPLC) was carried out on a
Thermo Scientific Accucore RP-MS solid core column
(100 × 3 mm, 2.6 µm particle size) at room temperature
using water and methanol as mobile phases (Figure 1).
The total runtime was 10 min. The mass spectrometer was
operated with an APCI source in negative ion mode. Data
was acquired in selected reaction monitoring (SRM) mode.
Figure 1. TurboFlow and LC method
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Figure 1: TurboFlow and LC method
Eluting
Loading
Loading:
A: water; B: methanol.
Eluting:
A: water; B: methanol.