Cornelia L. Boeser, Katherine L. Walker, Michael Belford, Mary Blackburn, Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA, USA, 95134
FAIMS PARAMETERS
FAIMS Settings
The FAIMS Pro Duo interface was operated in high resolution mode with an inner electrode
temperature of 80 oC and an outer electrode temperature of 100 oC to maximize selectivity. The
carrier gas was set to 4.6 L/min which is the default value for FAIMS operation on the TSQ Altis Plus
mass spectrometer.
FAIMS CV Optimization
Optimization of FAIMS CV values was performed on-line by injection using the new FAIMS CV
scanning option for SRM methods in the method editor (see Figure 2).
A coarse CV optimization injection was performed using a CV range of -25 to +25 V with a step size
of 4 V. Then, a fine optimization was performed using a CV range of +5 to +35 V with a step size of 2
V. After determining the optimum CVs for testosterone and testosterone-d3, the same fine
optimization was performed on a matrix blank. In Thermo Scientific FreeStyle 1.8 SP1, CV plots were
generated by using the ‘CV Merge’ function under ‘Auto Filter’ (see Figure 3) followed by the ‘CV Plot’
function. Sample and matrix plots were overlaid to determine the CV value which provides the best
S/N (see Figure 4).
ABSTRACT
Purpose: Improvement of LOQ for testosterone analysis by reduction of matrix background using
FAIMS technology.
Methods: Testosterone was analyzed with and without FAIMS technology at a flow rate of 250
uL/min using identical chromatographic conditions and mass spectrometer settings. FAIMS CV was
optimized on-line by injection against a matrix blank to find the optimum CV value.
Results: Matrix background was reduced significantly and LOQ of testosterone was improved 2-fold
from 1 pg/mL to 0.5 pg/mL.
INTRODUCTION
Testosterone has been banned in athletic competitions because of its performance enhancing
properties. Hence, quantitative determination of testosterone in humans has become important in the
field of sports doping. LC/MS assays developed for the analysis of testosterone often exhibit high
background signal which is difficult to eliminate by LC separation. This limits signal-to-noise ratio
(S/N) and ultimately limit of quantification (LOQ) of the assay. The Thermo Scientific™ FAIMS Pro
Duo interface spatially separates ions based on alternating high and low electric fields applied to a
set of cylindrical electrodes, enabling attenuation of matrix signal and increasing signal-to-noise. Here
we demonstrate improved LOQ for testosterone in human serum utilizing the Thermo Scientific™
FAIMS Pro Duo interface on the Thermo Scientific™ TSQ Altis™ Plus mass spectrometer (Figure 1).
MATERIALS AND METHODS
Sample Preparation
Testosterone was spiked into female human serum at concentrations ranging from 0.125 to 1000
pg/mL. Testosterone-d3 was added to the spiked serum at a concentration of 50 pg/mL. Liquid/liquid
extraction was performed on each sample using methyl tert-butyl ether (MTBE). After evaporation,
samples were reconstituted in 150 uL 70:30 water:methanol.
Test Method
A Thermo Scientific™ Vanquish™ Flex LC was used with a Thermo Scientific™ Accucore™
Vanquish™ C18+ column (P/N 27101-102130) at a flow rate of 250 uL/min. Methanol was used as
mobile phase B and 0.5 mM ammonium fluoride in water was used as mobile phase A. The injection
volume was 25 uL. Due to the limitation in sample volume, single injections were performed for each
calibrator level. The OptaMax NG ion source HESI sprayer was positioned at L (vertical alignment)
and 1 (front/back alignment) for both, runs with and without the FAIMS interface. Mass spectrometer
settings were identical for runs with and without the FAIMS interface. SRM table and mass
spectrometer settings are shown in Table 1 and 2, respectively.
CONCLUSIONS
Utilizing FAIMS technology as an additional dimension of separation can enhance LC/MS analysis
by selectively transmitting analyte ions through the electrodes while attenuating signal from matrix
and/or background ions.
FAIMS technology often provides improved signal-to-noise and LOQ, particularly when dealing
with complex matrices, such as human serum.
For quantitating testosterone in human serum, use of the FAIMS Pro Duo interface improved LOQ
2-fold from 1 pg/mL to 0.5 pg/mL
ACKNOWLEDGEMENTS
We would like to thank Prof. Jun Qu and his team at SUNY-Buffalo for developing strategies for CV
optimization used in this study.
TRADEMARKS/LICENSING
For Research Use Only. Not for use in diagnostic procedures. © 2021 Thermo Fisher Scientific
Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its
subsidiaries. This information is not intended to encourage use of these products in any manner that
might infringe the intellectual property rights of others.
PO66086 EN0921S
Quantitative Analysis of Testosterone from Human Serum using high-flow Liquid
Chromatography and FAIMS on a Triple Quadrupole Mass Spectrometer
Figure 5. Calibration curve for testosterone acquired with the FAIMS Pro Duo interface: (Left)
showing the full range up to 1000 pg/mL and (Right) showing the low range up to 10 pg/mL.
Data points lower than the LOQ (0.5 pg/mL) were excluded because of ion ratio failure.
Theoretical
Concentration
(pg/mL)
With the FAIMS Pro Duo Interface
Without the FAIMS Pro Duo Interface
Calculated
concentratio
n (ng/mL)
Accuracy
(%)
Ion
Ratio
(%)
Calculated
concentration
(ng/mL)
Accuracy
(%)
Ion
Ratio
(%)
0.5
0.50
-0.5
87.6
n.d.
n.d.
n.d.
1
1.03
2.9
74.8
1.01
1.7
77.6
2.5
2.48
-0.8
86.6
2.57
2.7
76.4
5
4.80
-3.9
85.4
4.21
-15.8
86.6
10
9.14
-8.6
91.0
10.3
3.4
79.6
50
48.9
-2.3
84.8
49.6
-0.8
86.8
100
101
1.3
83.5
102
2.4
87.0
500
524
4.7
84.0
511
2.2
87.0
1000
1071
7.1
84.6
1041
4.1
85.5
Figure 6. Calibration curve for testosterone acquired without the FAIMS Pro Duo interface:
(Left) showing the full range up to 1000 pg/mL and (Right) showing the low range up to 10
pg/mL. Data points lower than the LOQ (1 pg/mL) were excluded because there was no peak
distinguishable from background.
RESULTS
Calibration curves for both experiments, with and without the FAIMS interface, are shown in Figure 5
and Figure 6 respectively. Using FAIMS technology, the LOQ was improved from 1 pg/mL (without
FAIMS technology) to 0.5 pg/mL (with FAIMS technology). Acceptance criteria for LOQ were based
on accuracy (< 20%), linearity of the calibration curve (R > 0.99), and ion ratio of the confirming ion
(± 20%).
Figure 7 and 8 show a comparison of 0.5 and 1 pg/mL with (Figure 7) and without (Figure 8) FAIMS
technology. With the FAIMS interface, background is significantly reduced, which enables the
detection and reliable quantification of lower concentration levels.
Figure 1. FAIMS Pro Duo interface installed on a TSQ Altis Plus mass spectrometer.
LOQ
LOQ
Table 3. Back-calculated concentration, accuracy, and ion ratio of testosterone for each
concentration both with, and without the FAIMS Pro Duo interface
Figure 7. Comparison of the quan (left) and qual (right) peaks for testosterone with the FAIMS
Pro Duo interface (A) at a concentration of 0.5 pg/mL (at LOQ) and (B) 1 pg/mL.
(A)
(A)
(B)
(B)
MS parameters
Value
Positive Ion
3000 V
Sheath Gas
50 Arb
Aux Gas
13 Arb
Ion Transfer Tube Temperature
340 C
Vaporizer Temperature
350 C
Q1 resolution
0.7
Q3 resolution
0.7
CID gas
2 mTorr
Source Fragmentation
0
Compound
Precursor (m/z) Product (m/z) Collision Energy (V)
RF Lens (V)
Testosterone
289.267
96.967
22.74
61
289.267
108.967
24.97
61
Testosterone-d3
292.35
96.967
22.99
63
Table 1. SRM table for the analysis of testosterone on the TSQ Altis Plus mass spectrometer.
Figure 2. On-line CV Scanning workflow for SRM methods.
CV scan method in method editor
CV determination in FreeStyle
Output of CV optima as
.csv and reimport into
method editor
CV plot
0
20
40
60
80
100
5
10
15
20
25
30
35
R
el
at
iv
e A
bundanc
e
CV value
Matrix
Testosterone
Optimum CV value: 21 V
Figure 3. ‘CV Merge’ function under ‘Auto Filter’ in FreeStyle™ 1.8 SP1 which merges scan
filters with different CV values into one filter.
Figure 4. Overlaid CV plots of a testosterone sample and a matrix sample. The optimum CV
value which maximizes testosterone signal and minimizes matrix contribution is 21 V.
Results for back-calculated concentrations, accuracy, and ion ratios are summarized in Table 3. The
RSD of the internal standard across all runs was 7.3 % with the FAIMS Pro Duo interface and 9.4 %
without. The 2-fold improvement in LOQ observed with the FAIMS interface is attributed to reduction
of chemical background.
Data Analysis
Thermo Scientific™ TraceFinder™ 5.1 was used for quantitative data analysis. For FAIMS data, a
FAIMS raw file was associated with the Data Analysis Method in order to select scan filters with CV
information.
Table 2. SRM table for the analysis of testosterone on the TSQ Altis Plus mass spectrometer.
Figure 8. Comparison of the quan (left) and qual (right) peaks for testosterone without the
FAIMS Pro Duo interface (A) at 0.5 pg/mL (below LOQ) and (B) 1 pg/mL (at LOQ). At a
concentration of 0.5 pg/mL the confirming ion was not detected.
