Sensitive Quantification and Structural Elucidation of Steroids
App Note / Case Study
Last Updated: March 11, 2024
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Published: February 14, 2024
credit: iStock
Hormonal steroids regulate the majority of bodily functions, and the dysregulation of these molecules can play a role in the pathophysiology of human disease.
Early techniques to measure endogenous steroids, such as immunoassays, are problematic because they lack specificity for low-level steroids. Gas chromatography-mass spectrometry offers higher specificity and is sensitive to low-level steroids but requires extensive sample preparation.
This app note highlights why liquid chromatography-tandem mass spectrometry (LC-MS/MS), paired with electron-activated dissociation (EAD)-based fragmentation, has emerged as a key technique for steroid analysis.
Download this app note to discover:
- A method that enables robust, high-throughput analysis of hormonal steroids in human plasma
- Fragment ion generation that allows characterization not achievable using collision-induced dissociation
- How to improve analyte specificity during quantitative analysis
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Quantitative and qualitative analysis of steroids by highresolution mass spectrometry
Sensitive quantification and structural elucidation of steroids using the ZenoTOF 7600 system
Robert Proos1, Paul RS Baker1 Nicole Abbott2, Kasey Hill2, and Mitch Phelps2
1SCIEX, USA; 2 The Ohio State University, USA
This technical note demonstrates the ability of the SCIEX
ZenoTOF 7600 system to sensitively quantify steroids and to
qualitatively characterize their structures using electron-activated
dissociation (EAD)-based fragmentation. The high speed of the
MS/MS acquisition (133 Hz) mode enables high sample
throughput while maintaining good spectral quality. The
complimentary EAD fragmentation mode provides diagnostic
fragments to distinguish steroid isomers and isobars.
Hormonal steroids regulate most body functions and the
dysregulation of these molecules can play a role in the
pathophysiology of human disease. Early techniques to measure
endogenous steroids include immunoassay and gas
chromatography-mass spectrometry (GC-MS). Immunoassays
are problematic because they lack specificity for low-level
steroids due to interference from endogenous steroids present at
higher levels. In contrast, GC-MS offers higher specificity and is
sensitive to low-level steroids, however, it requires extensive
sample preparation via derivatization. More recently, steroid
analysis by liquid chromatography-tandem mass spectrometry
(LC-MS/MS) has emerged as a sensitive and specific technique
with a simplified sample preparation.
Here, the analysis of hormonal steroids by LC-MS/MS is
explored using the ZenoTOF 7600 system (Figure 1). The lower
limit of quantification (LLOQ) and the limit of detection (LOD)
were calculated for steroids and steroid levels were measured in
plasma samples. EAD-derived fragment ions were used to
structurally characterize the steroids of interest. EAD generated
structure-specific fragment ions that were sufficient to distinguish
steroid isomers and isobars during analysis without requiring
extensive chromatographic development.
Key features for steroid analysis using the
ZenoTOF 7600 system
• The method leveraged the speed and sensitivity of the
ZenoTOF 7600 system to enable robust, high-throughput
quantitative analysis of hormonal steroids
• Fragment ions generated by EAD provided structural details
that permitted characterization that cannot be achieved using
data generated by collision-induced dissociation (CID)
• The LLOQs calculated for all measured steroids by HRMS
were sufficient to accurately measure steroids in plasma
Figure 1. XIC of TOF MS (top) and TOF MS/MS (bottom) spectra using the MRMHR scan mode to detect steroids in NIST 1950 plasma extract.
Data presented were collected in the positive ion mode. Negative ion mode data are not shown. The y-axis for each spectrum is expanded 10-fold.
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Methods
Sample preparation: Stock solutions of analytes and internal
standards were diluted in PBS to generate standard curves using
technical replicates to determine the LOD and LLOQ of steroids
(Table 1). PBS was used as a matrix surrogate because plasma
contains significant amounts of endogenous steroids. Deuterated
internal standards were added to NIST 1950 plasma samples at
a final concentration of 1 ng/mL to quantify endogenous steroids.
For plasma samples and quality control (QC) samples, 5 µL of
the internal standard mixture was added to 200 µL of sample
with and 300 µL DI water. Samples were shaken @ 700 rpm for
5 minutes, and the protein was precipitated by adding 250 µL of
0.1M zinc sulfate and shaking @ 600 rpm for 5 minutes after
which 500 µL cold methanol was added. Samples were again
shaken at 500 rpm for 5 minutes, centrifuged at 2637 rcf for 10
minutes at room temperature, and the supernatant was collected
and loaded onto a preconditioned HLB SPE 30mg (30 µm)
plate. The plate was washed with DI water and steroids were
eluted with acetonitrile. The eluant was dried with N2, and
samples were reconstituted in 50 µL 50:50 methanol:water. The
samples were then analyzed by LC-MS/MS using the ZenoTOF
7600 system.
Chromatography: Prepared samples were separated by highperformance liquid chromatography (HPLC) using a Kinetex
biphenyl column from Phenomenex (2.6 µm particle size, 100 x
2.1 mm). Analytes were eluted from the column using a biphasic
gradient (Table 2). Mobile phase A was water with 0.2mM
ammonium fluoride and mobile phase B was methanol. The flow
Time % MPA % MPB
0 50 50
1 45 55
3.5 45 55
8.7 10 90
9 10 90
9.025 5 95
9.8 1 99
10.5 1 99
10.6 50 50
12 50 50
Table 2. Gradient conditions for steroid analysis.
Table 1. Standard curves for 17 steroid standards. Analytes were serially diluted to the final concentrations shown here. The concentrations for
internal standards were constant for each analyte. Concentrations are shown in ng/mL. A 15 µL aliquot of each sample was injected on column for
analysis.
Compound [Std 1] [Std 2] [Std 3] [Std 4] [Std 5] [Std 6] [Std 7] [Std 8] [Std 9] [Std 10]
11-Deoxycorticosterone 0.012 0.029 0.059 0.235 0.941 2.35 11.8 29.4 58.8 118
11-Deoxycortisol 0.012 0.029 0.059 0.118 0.294 0.88 2.35 5.88 11.8 58.8
17-Hydroxyprogesterone 0.029 0.059 0.118 0.588 1.176 2.35 4.71 9.41 17.6 29.4
5-α-Dihydrotestosterone 0.029 0.059 0.118 0.588 1.18 2.35 4.71 9.41 17.6 29.4
Aldosterone 0.029 0.059 0.118 0.235 0.588 1.18 2.35 5.88 8.82 11.8
Androstenedione 0.029 0.059 0.118 0.588 1.176 2.35 4.71 9.41 17.6 29.4
Androsterone 0.012 0.029 0.059 0.235 0.941 2.35 11.8 29.4 58.8 118
Corticosterone 0.012 0.029 0.059 0.235 0.941 2.35 11.8 29.4 58.8 118
Cortisol 1.18 2.94 5.88 11.8 58.8 118 147 294 882 1180
Cortisone 0.012 0.029 0.059 0.235 0.941 2.35 11.8 29.4 58.8 118
DHEA 0.012 0.029 0.059 0.235 0.941 2.35 11.8 29.4 58.8 118
DHEA-Sulfate 29.4 58.8 118 294 588 1180 1470 2350 4710 7060
Estradiol 0.029 0.059 0.118 0.235 0.588 1.18 2.35 5.88 8.82 11.8
Estrone 0.029 0.059 0.118 0.235 0.588 1.18 2.35 5.88 8.82 11.8
Etiocholanolone 0.029 0.059 0.118 0.235 0.588 1.18 2.35 5.88 8.82 11.8
Progesterone 0.012 0.029 0.059 0.235 0.941 2.35 11.8 29.4 58.8 118
Testosterone 0.012 0.029 0.059 0.118 0.294 0.882 2.350 5.88 11.8 58.8
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rate was 400 µL/min. The column temperature was held constant
at 50°C and the injection volume was 15 µL.
Mass spectrometry: Data were acquired with a ZenoTOF 7600
system using 2 methods. The LLOQ and LOD of various steroids
were determined using the scheduled high-resolution multiple
reaction monitoring scan mode (sMRMHR). The MRM transitions
used are shown in Tables 3 and 4 for positive and negative ion
modes, respectively. For positive ion mode analysis, CID and
EAD accumulation times were set at 10 and 35 ms, respectively.
In the negative ion mode, accumulation times were set at 25 and
35 ms, respectively. The compound-specific parameters used,
including collision energy (CE) and declustering potential (DP),
are listed in Tables 3 and 4. For DHEA-sulfate and estrone,
pseudo MRM transitions were used (precursor ion to precursor
ion) due to inefficient fragmentation. The impact of individual
ZenoTOF 7600 system parameters on compound analysis has
been previously described.1
Experiments to structurally characterize steroids via EAD-based
fragmentation were performed using a data-dependent
acquisition (DDA) scan mode. The TOF MS mass range was set
Table 3. MRM transitions and compound-specific parameter settings for steroids and their deuterated internal standards analyzed in the
positive ion mode. Isomeric standards with 1 or more identical MRM transitions are highlighted in the same color shade.
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to m/z 100–500 for positive ion mode and m/z 100–800 for
negative ion mode. The accumulation time was set to 100 ms.
The data-dependent MS/MS accumulation times used matched
those reported for the positive and negative polarity sMRMHR
experiments.
Data processing: All quantitative data were analyzed using the
Analytics module in SCIEX OS software. Qualitative data were
processed using the Explorer module in SCIEX OS software.
The Formula Finder and Chem Spyder were used to match
MS/MS spectra to chemical structures.
LLOQ and LOD determination for steroids on
the ZenoTOF 7600 system
Standard curves analyzed using the sMRMHR scan mode run
with CID-based fragmentation yielded LLOQ and LOD values for
all compounds tested (Table 5). DHEA-sulfate, estradiol and
estrone were measured in the negative ion mode and all other
compounds were measured in the positive ion mode.
Aldosterone had LOD and LLOQ values from both positive and
negative ion modes. These values were derived from linear
regression lines for each standard curve. The LOD and LLOQ
values calculated for each compound, except androsterone,
were equivalent to the lowest concentrations used in the
standard curve. The qualifying MRM transition (273.2/147.1) of
androsterone had an LLOQ around the fifth standard in the curve
listed in Table 1. In contrast, the quantitative MRM transition for
androsterone matched the lowest concentration of the standard
curve. The LLOQ values achieved therefore might have been
lower, had a greater concentration range been tested for the
standard curve. However, the limits achieved were sufficient to
enable accurate detection of all measured steroids in plasma.
Linear regression curves were generated for each steroid (Figure
2) and a linear dynamic range (LDR) > 5-orders of magnitude
was observed. The generated standard curves were within the
LDR, as exemplified by simultaneous runs of the standard
curves for 11-deoxycortisol and cortisol. The standard curve for
11-deoxycortisol included concentrations as low as 0.012 ng/mL,
whereas the standard curve for cortisol included concentrations
as high as 1180 ng/mL. This large difference in concentrations
spans a LDR of 5 orders of magnitude, from -2 to +3 log
concentration ratios (data not shown).
Quantification of steroids in NIST 1950
plasma
The pooled plasma standard from the National Institute of
Standards and Technology (NIST), NIST 1950, is an important
Table 5. LOD and LLOQ values for all analyzed steroids and their
concentrations in NIST 1950 plasma. The LOD and LLOQ values
have ng/mL units. The product ions (Q3 transitions) have m/z units. The
steroid concentrations have ng/mL units.
Compound Formula Mono M+H M+HH2O DP Q1 Q3 CE Q3 CE RT
Aldosterone C21H28O5 360.194 359.186 -70 359.190 189.092 -25 4.95
Aldosterone-D7 C21H21D7O5 367.238 366.23 -70 366.230 194.124 -25 4.93
DHEA-Sulfate C19H28O5S 368.166 367.158 -70 367.160 96.960 -50 367.16 -50 2.06
DHEA-Sulfate-D5 C19H23D5O5S 373.197 372.189 -70 372.190 97.966 -50 372.19 -50 2.01
Estradiol C18H24O2 272.178 271.17 -120 271.170 145.066 -55 5.14
Estradiol-D5 C18H19D5O2 277.209 276.201 -120 276.200 147.078 -55 5.11
Estrone C18H22O2 270.162 269.154 -120 269.150 269.155 -50 269.15 -50 6.71
Estrone-D4 C18H18D4O2 274.187 273.179 -120 273.180 147.078 -50 273.18 -50 6.67
Table 4. MRM transitions and compound-specific parameter settings for steroids and their deuterated internal standards analyzed in
the negative ion mode.
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reference standard for measuring endogenous metabolites. The
internal standard curves generated with primary reference and
internal standards in a PBS matrix surrogate were used to
measure the concentrations of the 17 monitored steroids (Table
5). A representative chromatogram showing the total ion
chromatogram (TIC) and the XIC for NIST 1950 is shown in
Figure 3. These data demonstrate that the ZenoTOF 7600
system has the requisite sensitivity and linear dynamic range to
measure these compounds in a biological matrix.
Structural characterization of steroid
standards using EAD-based fragmentation
Distinguishing structural isomers is a significant challenge in
bioanalysis. During MS/MS events, isolation of the precursor ion
in Q1 is a low-resolution event, regardless of whether the
instrument is nominal or accurate mass. Consequently, isobaric
molecules that have nearly the same mass can make it more
challenging to distinguish between isomers. This is further
complicated when analyzing complex matrices, such as plasma.
Traditional LC-MS/MS methodologies use CID to generate
fragments for quantification. If isomers and/or isobars interfere
with the analysis of CID-based fragments, then time-consuming
Figure 3. XIC of TOF MS (top) and TOF MS/MS (bottom) spectra using the MRMHR scan mode to detect steroid standards in PBS as a matrix
surrogate. Data presented were collected in the positive ion mode. The y-axis for each spectrum is expanded 10-fold.
Figure 2. Representative quantitative linear regression curves for steroids. The standard curve was designed to test the sensitivity of the assay
and linearity across a LDR spanning 4 orders of magnitude.
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chromatographic methods must be used to resolve these
compounds. However, the development of EAD using a tunable
electron emitter, such as that found on the ZenoTOF 7600
system, has improved the generation of unique diagnostic
fragment ions for singly charged molecules. The EAD-generated
fragments enable high compound specificity without the need for
extensive chromatographic method development.
EAD fragmentation can be used to distinguish between
molecules that appear identical with CID fragmentation. Figure 4
shows the identical CID product ion spectra for the isomers 11-
deoxycorticosterone and 17-hydroxyprogesterone, which both
have masses of m/z 331.227 (Table 3, highlighted in yellow).
MRM transitions are not sufficient to distinguish between the
CID-based spectra of these molecules. Therefore, the isomers
must first be chromatographically separated by HPLC.
In contrast, EAD-based fragmentation generated rich MS/MS
data for the same pair of isomers, without requiring an additional
chromatography step (Figure 5). EAD fragmentation results in
both homolytic (radical-based) and heterolytic (not radical-based)
scission events. Unlike CID, which uses thermal degradation to
cleave molecules at low-energy bonds, EAD cleaves functional
groups, ring structures and alkyl chains, independent of bond
energy. This results in a rich spectrum that can provide sufficient
structural information to fully characterize a molecule.
Figure 5 (top) shows 3 unique EAD-based fragments derived
from 11-deoxycorticosterone that were not present in the
spectrum for 17-hydroxyprogesterone (Figure 5, bottom).
Similarly, 2 unique fragments were found for 17-
Intensity Intensity
11-Deoxycortecosterone
17-Hydroxyprogesterone
EAD
EAD
269.1920
272.2142
257.1917
299.2020
287.2021
269.1920
Figure 5. EAD-based fragmentation spectra for 11-deoxycortecosterone (top) and 17-hydroxyprogesterone (bottom). Using a DDA approach,
TOF MS/MS scans were triggered using the MRMHR mode to produce high-quality full MS/MS spectra for each targeted molecule. The 2 structures
were very similar, however, EAD-based fragmentation generated unique, structurally diagnostic peaks that distinguished the isomers. MRM-based
experiments using EAD abbrogate the need to separate these compounds chromatographically.
Figure 4. CID-based fragmentation MS/MS spectra for isomeric
11-deoxycorticosterone (top) and 17-hydroxyprogesterone
(bottom). These 2 steroid positional isomers have identical CID-based
MS/MS spectra. The resolution of these molecules relies on
chromatographic separation.
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hydroxyprogesterone. These product ions can be used in
sMRMHR experiments using EAD-based fragmentation to
increase the specificity of the assay by distinguishing between
these 2 isomers.
This pattern of results was not unique to the 11-
deoxycorticosterone and 17-hydroxyprogesterone isomers.
Similar data were obtained for the other 3 sets of isomers (Table
3, highlighted in blue, green and orange) and structurally specific
EAD-based fragment ions were found that could distinguish each
compound (data not shown).
Conclusions
• The method leverages the speed and sensitivity of the
ZenoTOF 7600 system to enable robust, high-throughput
analysis of hormonal steroids in human plasma
• Fragment ions generated by EAD provide structural details
that allow characterization that cannot be achieved using data
collected by CID
• The simultaneous use of CID- and EAD-based fragmentation
during quantitative analysis supports high-throughput analysis
while improving analyte specificity
References:
1. Baker, PRS and Proos R. Untargeted data-dependent
acquisition (DDA) metabolomics analysis using the
ZenoTOF 7600 system. https://sciex.com/technotes/life-science-research/metabolomics/untargeteddata-dependent-acquisition--dda--metabolomics-analysi
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