Page 55 - CRFTApplication Compendium_090613

Selective Testosterone Analysis in
Human Serum by LC-FAIMS-MS/MS
Jonathan McNally and Michael Belford, Thermo Fisher Scientific, San Jose, CA
James Kapron, Thermo Fisher Scientific, Ottawa, ON, Canada
Application
Note: 446
Key Words
• TSQ Quantum
Ultra
• Clinical analysis
• High-throughput
• Selectivity
Overview
Current high throughput clinical assays utilizing triple
quadrupole mass spectrometry for the quantitation of
testosterone can be further enhanced through the use of
FAIMS (high-Field Asymmetric waveform Ion Mobility
Spectrometry) coupled to a TSQ Quantum triple quadru-
pole mass spectrometer. This application note describes
how a FAIMS-enabled TSQ Quantum improves the per-
formance of a testosterone assay.
Introduction
Testosterone is the androgenic hormone primarily respon-
sible for normal growth and development of male repro-
ductive organs. Although testosterone production declines
naturally with age, testosterone production may be com-
promised by diseased or damaged organs.
Women biosynthesize very low levels of testosterone,
which makes quantitation extremely difficult. Estrogen
replacement therapy may further reduce testosterone pro-
duction, resulting in additional complications in its quan-
titation. In addition, endogenous interferences may
prevent accurate and precise testosterone measurement.
In this study, an LC-MS/MS method was used together
with the selectivity offered by FAIMS to quantify testos-
terone in human serum. FAIMS acts to remove chemical
background and endogenous interferences resulting in
more accurate and precise determinations for clinical
samples than LC-MS/MS alone.
Experimental Conditions
Chemicals and Reagents
Testosterone and testosterone-d3 (internal standard-IS)
were purchased from Sigma-Aldrich (St. Louis, MO).
HPLC grade methanol and formic acid were acquired
from Burdick and Jackson (Muskegon, MI). All chemicals
were used as received.
Sample preparation: Stripped human serum (Golden
West Biologicals, Temecula, CA) was fortified with testos-
terone at the following concentrations: 2.5, 5, 10, 25, 50,
100, 250, 500, 1000, and 2500 pg/mL. Internal standard
in 5% formic acid was added to a final concentration of
500 pg/mL. No further sample preparation was required.
Sample analysis: LC-MS/MS analyses were performed
on a Thermo Scientific Surveyor LC system. The method
used mobile phases A (0.1% formic acid in water) and B
(0.1% formic acid in methanol) at a flow rate of 0.5
mL/min. Serum samples (90 µL) were injected onto an LC-
MS/MS extraction column. The analyte was back-flushed
to the 2.1 × 50 mm, 3 µ, Thermo Scientific Hypersil GOLD
analytical column. The entire LC effluent from the sample
injections was directed to the Ion Max source on a Thermo
Scientific TSQ Quantum Ultra.
Additional gas-phase separation prior to entry of ions
into the mass spectrometer was achieved by including
FAIMS in the analysis.
FAIMS Conditions
Dispersion voltage –5000 V
Outer bias voltage 35 V
Compensation voltage –12.5 V
Temperature (inner electrode) 60 °C
Temperature (outer electrode) 60 °C
FAIMS gas composition 50% He in N 2
FAIMS gas flow rate 3.8 L/min
MS Conditions
Ionization mode
and source Positive APCI
Spray current 1.0 µA
Vaporizer temperature 400°C
Sheath gas 35
Transfer tube
temperature 250 °C
Transfer tube offset 35 V
Tube lens offset 100 V
Collision energy 22 eV
Scan time 50 ms
Q1 Resolution 0.7 Da FWHM
Q3 Resolution 0.7 Da FWHM
Testosterone m/z 289.2 m/z 97.1, 109.1
Testosterone-d 3 m/z 292.2 m/z 97.1, 109.1
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