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Fast and Sensitive LC-APCI-MS/MS
Quantitative Analysis of Estrone and Estradiol
in Serum without Chemical Derivatization
Xiang He and Marta Kozak; Thermo Fisher Scientific, San Jose, CA
Application
Note: 530
Key Words
• TSQ Vantage
• Accela UHPLC
• Clinical Research
Introduction
In the clinical research setting, quantitative measurements
of estrone (E1) and estradiol (E2) in serum typically have
been done with immunoassay or liquid chromatography-
tandem mass spectrometry (LC-MS/MS). LC-MS/MS
is preferred over immunoassay and other analytical
techniques because of its high sensitivity.
E1 and E2 are usually chemically derivatized before
they are detected by mass spectrometry for enhanced
sensitivity. The derivatization step extends the sample
preparation procedure and usually involves chemicals/
reagents that might compromise the performance of the
mass spectrometer in the long term.
Goal
To develop and validate a simple, fast and sensitive
analytical method for measuring E1 and E2 in serum or
plasma by LC-APCI-MS/MS.
Methods
Sample Preparation
Serum was spiked with internal standard (IS, deuterated
E2) and underwent liquid-liquid extraction (LLE) with
methyl tert-butyl ether (MTBE). After extraction, the
MTBE layer was dried under nitrogen and re-suspended
with 60% methanol. The reconstituted sample was
centrifuged to remove particulates and the supernatant was
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 Accela UHPLC system.
UHPLC was carried out on a Thermo Scientific Hypersil
GOLD column (150 × 2.1 mm, 3 µm) at room temperature
using water and methanol as mobile phases. The total
LC run time was 6 minutes. The mass spectrometer was
operated with an atmospheric pressure chemical ionization
(APCI) source in negative ion mode. Data was acquired in
selected reaction monitoring (SRM) mode.
Validation
The validation procedure included tests for 1) recovery of
sample preparation; 2) calibration range; 3) lower limit of
quantitation (LLOQ), dynamic range, accuracy;
4) precision; 5) ion suppression; and 6) carryover.
Results and Discussion
Sample Preparation
LLE was used to extract E1 and E2 from serum/plasma
and was found to be efficient. MTBE was selected as the
extraction solvent for its excellent recovery and ease of
handling.
Validation
1. Recovery for LLE Sample Preparation
The absolute recovery of E1, E2 and their internal
standard from liquid-liquid extraction ranged from
70% – 115% (n=4).
2. Calibration Range
Calibration curves (Figures 1 and 2) using calibrators in
charcoal stripped serum (CSS) showed excellent linearity
(R
2
> 0.998) between 5 and 1000 pg/mL.
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