toxics

Article
Determination of 19 Steroid Hormones in Human Serum and
Urine Using Liquid Chromatography-Tandem
Mass Spectrometry
Zhong-Min Li 1,2 and Kurunthachalam Kannan 1,2, *

1 Department of Pediatrics, New York University Grossman School of Medicine, New York, NY 10016, USA
2 Department of Environmental Medicine, New York University Grossman School of Medicine,
New York, NY 10016, USA
* Correspondence: [email protected]; Tel.: +1-(212)-263-1546

Abstract: This paper describes a methodology for simultaneous determination of 19 steroid hormones,
viz. estrone, estradiol, estriol, testosterone, 5α-dihydrotestosterone, androstenedione, androstenediol,
dehydroepiandrosterone, progesterone, pregnenolone, 17α-OH-progesterone, 17α-OH-pregnenolone,
cortisone, cortisol, 11-deoxycortisol, 11-deoxycorticosterone, 11-dehydrocorticosterone, aldosterone,
and corticosterone, in 500-µL of urine or serum/plasma. The method was optimized using iso-
topically labeled internal standards and liquid-liquid extraction followed by detection using liquid
chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS). Dansylation of
estrogens significantly improved their sensitivities (~11- to 23-fold) and chromatographic separation.
The respective limit of detection (LOD) and limit of quantification (LOQ) of all analytes were 0.04–0.28
and 0.14–0.92 ng/mL in human urine, and 0.11–0.35 and 0.38–1.18 ng/mL in human serum/plasma.
Recoveries of all analytes (except for progesterone) fortified at 10, 20, and 200 ng/mL in urine and
Citation: Li, Z.-M.; Kannan, K.
serum were 80–120%, with standard deviations ranging from 0 to 17.3%. Repeated analysis of
Determination of 19 Steroid
Hormones in Human Serum and
similarly fortified urine and serum samples yielded intra-day and inter-day variations of 0–21.7%
Urine Using Liquid and 0.16–11.5%, respectively. All analytes except cortisone exhibited weak matrix effects in urine
Chromatography-Tandem Mass and serum (−13.9–18.2%). The method was further validated through the analysis of the National
Spectrometry. Toxics 2022, 10, 687. Institute of Standards and Technology (NIST) plasma Standard Reference Material (SRM1950) with
https://doi.org/10.3390/ certified concentrations for cortisol, progesterone, and testosterone (coefficient of variation: 3–11%).
toxics10110687 The developed method was applied in the analysis of urine samples from 20 volunteers, which re-
Academic Editors: Roel Vermeulen,
vealed the occurrence of 16 analytes with detection frequencies (DFs) > 80%. Furthermore, 15 analytes
Lauren Petrick, Maaike van Gerwen were found in plasma SRM1950, indicating the feasibility of our method in the analysis of steroid
and Kelvin S. Y. Leung hormones in urine and serum/plasma. This method will facilitate analysis of steroid hormones in
population-based biomonitoring studies.
Received: 10 October 2022
Accepted: 9 November 2022
Keywords: steroid hormones; estrogens; androgens; progestogens; corticosteroids; urine; serum
Published: 12 November 2022

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations.
Steroid hormones are cholesterol derivatives that play critical roles in regulating water
and salt homeostasis, metabolism, stress response, and in initiating and maintaining sexual
differentiation and reproduction [1]. Steroid hormones are synthesized through a cascade-
Copyright: © 2022 by the authors.
like pathway in the adrenal cortex, the gonads and the placenta and are released into the
Licensee MDPI, Basel, Switzerland. blood stream to act in both peripheral target tissues and the central nervous system. The
This article is an open access article homeostasis of steroid hormones is regulated through hypothalamus-pituitary-gonadal
distributed under the terms and (HPG) and hypothalamus-pituitary-adrenal (HPA) axes [2,3], which consist stimulatory
conditions of the Creative Commons hormones and feedback loops. Steroid hormones can be classified as estrogens, androgens,
Attribution (CC BY) license (https:// progestogens, and corticosteroids (Figure 1), according to their structures and genomic
creativecommons.org/licenses/by/ receptors to which they bind [4]. In general, estrogens (18 carbons with an aromatic ring) are
4.0/). female reproductive hormones, androgens (19 carbons) are male reproductive hormones,

Toxics 2022, 10, 687. https://doi.org/10.3390/toxics10110687 https://www.mdpi.com/journal/toxics

Toxics 2022, 10, x FOR PEER REVIEW 2 of 15

Toxics 2022, 10, 687 2 of 15

genomic receptors to which they bind [4]. In general, estrogens (18 carbons with an aro-
matic ring) are female reproductive hormones, androgens (19 carbons) are male reproduc-
tive hormones,
progestogens (21progestogens
carbons) are (21 carbons)hormones,
pregnancy are pregnancy hormones, and(21
and corticosteroids corticosteroids
carbons) are
(21 carbons) are stress
stress hormones [5]. hormones [5].

Figure 1.
Figure 1. Molecular
Molecular structures
structures and
and metabolic
metabolic pathway
pathway of
of the
the 19
19 steroid
steroid hormones
hormones investigated
investigated in
in
this study.
this study.

The lipophilic
The lipophilic steroid
steroid hormones
hormones undergo
undergo phase
phase II and
and phase
phase II
II metabolism,
metabolism, and and are
are
excreted mainly through
through urine
urine as
as glucuronides,
glucuronides, sulfates,
sulfates, diglucuronides,
diglucuronides, disulfates,
disulfates, and
and
sulfoglucuronides[1,6].
sulfoglucuronides [1,6].Perturbation
Perturbationininsteroid
steroid hormone
hormone homeostasis
homeostasis in blood,
in blood, urine,
urine, sa-
saliva,
liva, hair
and and has
hairbeen
has been measured
measured in investigations
in investigations focused
focused on cancer
on cancer [7–11],[7–11],
stressstress
[12], [12],
and
and endocrine-disruption
endocrine-disruption by environmental
by environmental chemicals
chemicals suchsuch as bisphenols
as bisphenols [13], [13], phthalates
phthalates [14]
[14] triclosan
and and triclosan
[13]. [13].
methods of
Traditional methods of analysis
analysis of
of steroid
steroid hormones
hormones in in human
human specimens
specimens (such (such asas
serum) are
serum) areradioimmunoassays
radioimmunoassays (RIAs)
(RIAs) and enzyme-linked
and enzyme-linked immunosorbent
immunosorbent assays
assays (ELISAs).
These methods
(ELISAs). Theseare highly sensitive,
methods are highlybut lack selectivity
sensitive, but lackdue to nonspecific
selectivity due to antigen-antibody
nonspecific anti-
interactions
gen-antibody [15]. Besides, RIAs
interactions and ELISAs
[15]. Besides, RIAs usually target usually
and ELISAs one analyte
target(i.e.,
one hormone) per
analyte (i.e.,
assay.
hormone)Methods basedMethods
per assay. on mass based
spectrometry
on massoffer improved offer
spectrometry robustness,
improved specificity and
robustness,
accuracy,
specificityand andhave been routinely
accuracy, and have used
beenforroutinely
steroid hormone
used foranalysis
steroidinhormone
recent years. For in-
analysis in
stance,
recent gas
years.chromatography-mass spectrometry (GC-MS) spectrometry
For instance, gas chromatography-mass methods provide high sensitivity,
(GC-MS) methods
selectivity
provide high andsensitivity,
accuracy. However,
selectivitydue
andtoaccuracy.
the low volatilities
However,of steroid
due to thehormones, analysis
low volatilities of
by GC-MS requires derivatization, a laborious and time consuming
steroid hormones, analysis by GC-MS requires derivatization, a laborious and time step [16,17]. To over-
con-
come this problem, studies have applied liquid chromatography coupled with tandem
mass spectrometry (LC-MS/MS) [18–22]. Nevertheless, challenges exist in simultaneous
measurement of ultra-trace levels of several steroid hormones in serum or urine. Estrogens
Toxics 2022, 10, 687 3 of 15

exhibit low sensitivities due to their low ionization efficiencies [23], leading to difficulties
in analysis at trace levels of these hormones [24]. Besides, many steroid hormones have
similar chemical structures and molecular fragmentation patterns, which make their mass
spectrometric identification difficult. The loss of one or two water moiety from the molecule
is a common mass fragmentation feature observed for steroid hormones. Several reported
earlier studies entailed long time (e.g., 40 min) for chromatographic separation [5,19], which
can hamper high throughput analysis in large-scale population-based studies.
We describe a method for sensitive and selective determination of four classes of
steroid hormones simultaneously in human urine and serum/plasma. The goals of this
study were: (1) to develop a LC-MS/MS method for the determination of physiologi-
cally relevant concentrations of 19 steroid hormones in a single extraction and injection;
(2) to evaluate and validate the method for sensitivity, accuracy and precision in human
urine and serum/plasma; and (3) to assess the feasibility of the method in measuring
steroid hormones in urine samples from the general population and in pooled human
serum/plasma. Nineteen steroid hormones, comprising 3 estrogens (estrone, estradiol and
estriol), 5 androgens (testosterone, 5α-dihydrotestosterone, androstenedione, androstene-
diol, and dehydroepiandrosterone (DEHA)), 4 progestogens (progesterone, pregnenolone,
17α-OH-progesterone, and 17α-OH-pregnenolone), and 7 corticosteroids (cortisone, corti-
sol, 11-deoxycortisol, aldosterone, 11-deoxycorticosterone, 11-dehydrocorticosterone, and
corticosterone) were the target analytes.

2. Materials and Methods

2.1. Chemicals and Reagents
The chemical structures of the 19 steroid hormones investigated in this study are shown
in Figure 1. Individual certified stock solutions of cortisone, 11-deoxycortisol, aldosterone,
corticosterone, progesterone, 17α-OH-progesterone, pregnenolone, estrone, estradiol, testos-
terone, dehydroepiandrosterone (DEHA), 5α-dihydrotestosterone, 17α-OH-pregnenolone,
11-dehydrocorticosterone, androstenediol, 13 C3 -cortisone, 13 C3 -cortisol, 13 C3 -11-deoxycortisol,
D4 -aldosterone, D4 -corticosterone, 13 C3 -progesterone, 13 C3 -17α-OH-progesterone, 13 C2 -D2 -
pregnenolone, 13 C3 -estrone, 13 C2 -estradiol, 13 C3 -estriol, 13 C3 -testosterone, 13 C3 -DEHA, 13 C3 -
5α-OH-dihydrotestosterone, and 13 C2 -D2 -17α-OH-pregnenolone (100 µg/mL) with purities
of 95–98% were purchased from Cambridge Isotope Laboratories (Andover, MA, USA).
Neat cortisol and estriol (purity: ≥95%) were obtained from Cambridge Isotope Laborato-
ries (Andover, MA, USA). Individual stock solutions of cortisol and estriol (100 µg/mL)
were prepared in methanol (MeOH). Individual certified stock solutions of 11-deoxycor-
ticosterone, androstenedione and 13 C3 -11-deoxycorticosterone (100 µg/mL) with purities
of ≥98% were purchased from Sigma-Aldrich (St. Louis, MO, USA). All stock solutions
were stored at −20 ◦ C. Working solutions of analytical standards were diluted from stock
solutions using acetonitrile (ACN).
Synthetic urine was purchased from Cerilliant (Round Rock, TX, USA). Pooled hu-
man serum was from Sigma-Aldrich (St. Louis, MO, USA). Plasma Standard Reference
Material (SRM1950) was purchased from the National Institute for Standards and Tech-
nology (NIST; Gaithersburg, MD, USA) through Sigma-Aldrich (St. Louis, MO, USA).
β-glucuronidase/arylsulfatase enzyme (ALS; β-glucuronidase: ~100,000 units/mL; aryl-
sulfatase: ~47,500 units/mL) from Helix pomatia was obtained from Roche Life Science
through Sigma-Aldrich (St. Louis, MO, USA). L -Ascorbic acid, dansyl chloride (DC), formic
acid (88%), and ammonium acetate (NH4 Ac) of analytical grade were from Sigma-Aldrich
(St. Louis, MO, USA). Water, ACN, methyl tert-butyl ether (MTBE), and ethyl acetate
(EtAc) of LC-MS grade were from Fisher Scientific (Waltham, MA, USA). Bond Elut C18
(60 mg/3 mL), Bond Elut Plexa (60 mg/3 mL), and Bond Elut NEXUS (60 mg/3 mL) solid
phase extraction (SPE) cartridges were obtained from Agilent Technologies (Santa Clara,
CA, USA).
Toxics 2022, 10, 687 4 of 15

2.2. Sample Collection

Spot urine samples were collected randomly in 50-mL polypropylene (PP) tubes from
11 male (age: 18–64 years, average 38.0 years) and 9 female (age: 15–59 years, average
33.3 years) volunteers from New York City during May–June 2022. Immediately after col-
lection, samples were stored at −20 ◦ C until analysis. The urine samples were deidentified
and therefore fell under the ‘exempt’ category of the New York University Institutional
Review Board.

2.3. Preparation of Urine and Serum Samples

A 500-µL aliquot of urine was pipetted into a 15-mL borosilicate glass culture tube.
Synthetic urine and pooled human urine fortified with target analytes (at concentrations of
10, 20, and 200 ng/mL) were used for method development and validation. Then, 2.5–12.5 ng
of isotopically labelled internal standards were spiked into each sample, and 500 µL of 1 M
NH4 Ac buffer (pH 5.5) containing 2.5 mg of L -ascorbic acid and 20 µL of ALS enzyme
(2000 units) were added. After gentle mixing, the samples were incubated overnight (~15 h)
at 37 ◦ C by shaking at 100 rpm (Jeio Tech Co., Seoul, South Korea). Thereafter, 1 mL of HPLC-
grade water was added to each sample, followed by the addition of 4 mL MTBE/EtAc (5:1,
v/v). After ultra-sonication for 15 min and shaking for 30 min in a reciprocating shaker, the
samples were subjected to centrifugation at 3000 rpm for 5 min, and the supernatant was
transferred into a new glass tube. The liquid-liquid extraction (LLE) was repeated twice
with 3 mL of MTBE/EtAc (5:1, v/v). The combined extracts were subsequently evaporated
to dryness under N2 at 25 ◦ C. Estrogens were then selectively derivatized by the addition
of 125 µL of sodium bicarbonate buffer (0.1 M; pH 9.0) and 125 µL of dansyl chloride
(1 mg/mL in acetone), vortexed vigorously (~30 s), and the sample tubes were immediately
kept at 60 ◦ C (on a hot plate) for 5 min. The extracts were evaporated to dryness under
N2 at 25 ◦ C, reconstituted in 250 µL MeOH, and transferred into amber glass vials for
LC-MS/MS analysis.
Serum samples (500 µL) were processed by following the procedure described above,
without enzymatic hydrolysis. Pooled human serum fortified with target analytes (at concen-
trations of 10, 20, and 200 ng/mL) was used for method development and validation.

2.4. LC-MS/MS Analysis

The quantification of target analytes was performed on an ABSciex 5500+ Q-trap mass
spectrometer (Framingham, MA, USA) coupled to an ExionLC HPLC (SCIEX, Redwood City,
CA, USA). Analytes were separated on an Eclipse Plus C18 RRHD column (150 × 2.1 mm,
1.8 µm; Agilent Technologies, Santa Clara, CA, USA) coupled to a BetaSil C18 guard column
(20 × 2.1 mm, 5 µm; Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase,
maintained at a flow rate of 0.2 mL/min, consisted water (A) and ACN (B) each containing
0.1% formic acid (v/v). The following gradient program was applied: hold at 10% B for
0.5 min, linear ramp to 40% B over 0.5 min, linear ramp to 70% B over 9 min, then linear
ramp to 95% B over 0.5 min, hold at 95% B for 4.5 min, then return to initial conditions
over 0.5 min, and equilibrate for 1.5 min prior to the next injection. The mobile phase
flow was diverted to waste during the first 3.5 min after sample injection. The column
was maintained at room temperature; the autosampler was kept at 15 ◦ C; and the injection
volume was 10 µL.
All analytes were measured using electrospray ionization (ESI) multiple reaction mon-
itoring (MRM) in positive-ion mode. The optimized MRM parameters including collision
energy (CE), declustering potential (DP), collision cell exit potential (CXP), entrance po-
tential (EP), and dwell time are given in Table 1 and Table S1 (Supplementary Material).
The IonSpray voltage was 5.5 kV; the ion source temperature was 500 ◦ C; the curtain gas
and collision gas flow rates were set at 20 and 9 psi, respectively; and the ion source gases 1
and 2 were set at 70 and 60 psi, respectively. Analyst software v1.7.2 (ABSciex, Framingham,
MA, USA) was used for data analysis.
Toxics 2022, 10, 687 5 of 15

Table 1. Target analytes (19 steroid hormones), isotopically labeled internal standards, and their
MRM parameters. MRM parameters include precursor ion (Q1), product ion (Q3), declustering
potential (DP), entrance potential (EP), collision energy (CE), collision cell exit potential (CXP), and
dwell time.

Name CAS Q1 (m/z) Q3 (m/z) DP (V) CE (V) EP (V) CXP (V) Dwell (ms)
Target analytes
DC-Estrone 504 171 125 35 10 15 30
DC-Estradiol 506 171 125 35 10 15 30
DC-Estriol 522 171 125 35 10 15 30
Testosterone 58-22-0 289 97 100 29 10 12 30
5α-Dihydrotestosterone 521-18-6 291 255 100 22 10 14 30
Androstenedione 63-05-8 287 97 172 25 10 14 30
Androstenediol 521-17-5 273 255 93 17 10 8 30
DEHA 53-43-0 271 253 80 23 10 12 30
Progesterone 57-83-0 315 97 130 23 10 10 30
Pregnenolone 145-13-1 317 299 98 13 10 16 30
17α-OH-Progesterone 68-96-2 331 109 114 39 10 6 30
17α-OH-Pregnenolone 387-79-1 333 297 80 23 10 18 30
Cortisone 53-06-5 361 163 152 31 10 20 30
Cortisol 50-23-7 363 121 144 29 10 12 30
11-Deoxycortisol 152-58-9 347 97 161 27 10 10 30
11-Deoxycorticosterone 64-85-7 331 97 160 27 10 16 30
11-Dehydrocorticosterone 72-23-1 331 97 168 26 10 11 30
Corticosterone 50-22-6 347 329 100 23 10 18 30
Aldosterone 52-39-1 361 343 120 24 10 12 30
Internal standards
DC-13 C3 -Estrone 507 171 125 35 10 15 30
DC-13 C2 -Estradiol 508 171 125 35 10 15 30
DC-13 C3 -Estriol 525 171 125 35 10 15 30
13 C -Testosterone 292 100 120 27 10 14 30
3
13 C -5α-Dihydrotestosterone 294 258 130 21 10 12 30
3
13 C -Dehydroepiandrosterone 274 256 125 14 10 14 30
3
13 C -Progesterone 318 100 170 30 10 6 30
3
13 C -D -Pregnenolone 321 303 110 11 10 22 30
2 2
13 C -17α-OH-Progesterone 334 100 138 27 10 12 30
3
13 C -D -17α-OH-Pregnenolone 319 301 90 15 10 16 30
2 2
13 C -Cortisol 366 124 152 30 10 22 30
3
13 C -11-Deoxycortisol 350 112 132 32 10 17 30
3
13 C -11-Deoxycorticosterone 334 100 154 27 10 18 30
3
D4 -Corticosterone 351 333 126 17 10 18 30
D4 -Aldosterone 365 347 100 26 10 24 30
Abbreviations: DC-Estrone, dansylated estrogen; DC-Estradiol, dansylated estradiol; DC-Estriol, dansylated
estriol; DEHA, dehydroepiandrosterone.

2.5. Method Validation

Calibration curves of all analytes were constructed both in solvent and in fortified
urine and serum matrices. Calibration standards were prepared in ACN at concentrations
ranging from 0.05 to 2000 ng/mL, with 10–50 ng/mL of isotopically labelled internal
standards, and were derivatized by following the procedure described above. The matrix-
matched calibration curves were obtained by spiking all analytes at different concentrations
(0.05–2000 ng/mL) in urine and serum extracts.
Matrix effects were evaluated by calculating the percentage of signal enhancement or
suppression, as shown in Equation (1):
 
A
Matrix effect = − 1 × 100% (1)
B

where A and B are the slopes of the matrix-matched calibration curve, and calibration curve
from pure solvent, respectively.
Accuracy of the method was determined as the recoveries of analytes spiked at three
different concentrations (10, 20 and 200 ng/mL) in both pooled urine and serum. Precision
was assessed as intra-day and inter-day variations in measured concentrations in three
Toxics 2022, 10, 687 6 of 15

pooled urine or serum samples fortified at 10, 20 and 200 ng/mL and was calculated as
the coefficient of variation (CV%). The inter-day CV was measured by repeated injection
of fortified samples on three different days. Accuracy of the method was further assessed
through the analysis of NIST SRM1950 plasma, which has certified reference values for
cortisol, progesterone and testosterone.
To calculate the method limit of detection (LOD) and limit of quantification (LOQ) of
steroid hormones in urine, six replicates of synthetic urine were fortified with each target
analyte at 1 ng/mL, a concentration that yielded signal-to-noise (S/N) ratios of 6.4–62.4.
LOD and LOQ of target analytes in urine were calculated as 3 and 10 times the standard
deviation (SD), respectively. Similarly, LOD and LOQ in serum were calculated as 3 and
10 times the SD of concentrations, respectively, measured in pooled human serum spiked
at 1 ng/mL.

2.6. Quality Assurance and Quality Control

Quality control (QC) samples include procedural blanks (LC-MS grade water instead
of urine or serum), matrix blanks (pooled urine or serum), and matrix spiked samples
(analytes fortified in pooled urine or serum at three different concentrations: 10, 20 and
200 ng/mL). The NIST SRM sample was also included in the analysis of hormones in plasma.

3. Results and Discussion

3.1. Chromatography and Mass Spectrometry
The development of the method started with optimization of MS/MS parameters
for 19 target analytes, through the infusion of a standard solution (200 ng/mL in MeOH
containing 0.2% formic acid) directly into the mass spectrometer. Addition of formic acid to
the standard solution (as an additive) over acetic acid yielded better signal intensity. ACN,
as the solvent, yielded lower backpressure in comparison to MeOH.
Chromatographic separation of steroid hormones in urine or serum is critical because
several of these analytes exhibit the same mass spectrometric MRM transitions (Table 1).
Few earlier studies employed lengthy chromatographic separation time to isolate and
resolve individual analytes [5,19]. We compared the performance of several reversed-phase
LC columns and found that Eclipse Plus C18 RRHD column (150 × 2.1 mm, 1.8 µm; Agilent
Technologies, Santa Clara, CA, USA) provided good peak shape and chromatographic sepa-
ration for most of the analytes within a run time of 10 min (Figure S1). Nevertheless, several
important biomarkers were not resolved chromatographically. Estriol and aldosterone, as
well as estrone and DEHA co-eluted at the same retention times. Furthermore, estrogens
exhibited low signal intensities (Figure 2) due to their low ionization efficiencies. To im-
prove selectivity and sensitivity of estrogens, we applied an estrogen-specific derivatization
step using dansyl chloride, as reported earlier [18,19,25]. Following dansylation, the signal
intensities of estrone, estradiol, and estriol improved significantly with an enhancement
in S/N by 23, 16 and 11 times, respectively, in comparison to intensities obtained prior to
derivatization. These results are consistent with those reported earlier [26]. Furthermore, the
retention times of dansyl-estrone (DC-estrone), DC-estradiol, and DC-estriol were different
from all other analytes, indicating improved selectivity (Figures 2 and 3). Nevertheless,
androstenedione and 17α-OH-progesterone co-eluted, which, however, was deemed ac-
ceptable because of their different MRM transitions (Table 1). Our method, with a total
run time of 17 min, was faster than the previous methods [5,19], a feature favorable for
large-scale human biomonitoring studies.
 obtained prior to derivatization. These results are consistent with those reported earlier
[26]. Furthermore, the retention times of dansyl-estrone (DC-estrone), DC-estradiol, and
DC-estriol were different from all other analytes, indicating improved selectivity (Figures
2 and 3). Nevertheless, androstenedione and 17α-OH-progesterone co-eluted, which,
Toxics 2022, 10, 687 however, was deemed acceptable because of their different MRM transitions (Table 7 of1).
15
Our method, with a total run time of 17 min, was faster than the previous methods [5,19],
a feature favorable for large-scale human biomonitoring studies.

Figure 2.
Toxics 2022, 10, x FOR PEER REVIEWFigure 2. Comparison
ComparisonofofLC-MS/MS
LC-MS/MSresponses of estrone,
responses estradiol,
of estrone, andand
estradiol, estriol before
estriol and after
before 8 of
and dan-
15
after
sylation. Concentrations
dansylation. of the
Concentrations analytes
of the were
analytes 1 ng/mL;
were Injection
1 ng/mL; volume
Injection waswas
volume 10 µL.
10 µL.

Figure
Figure 3.
3. Representative
RepresentativeLC-MS/MS
LC-MS/MS chromatograms
chromatograms of of
19 19
steroid hormones
steroid in solvent.
hormones Concentra-
in solvent. Concen-
tions of the target analytes were 100 ng/mL; Injection volume was 10 µL; estrone, estradiol, and
trations of the target analytes were 100 ng/mL; Injection volume was 10 µL; estrone, estradiol, and
estriol were derivatized using dansyl chloride. Estrone and estradiol were shown separately because
estriol were derivatized using dansyl chloride. Estrone and estradiol were shown separately because
of their significantly higher intensities than the other analytes.
of their significantly higher intensities than the other analytes.
3.2. Optimization of Sample Purification
Given the lipophilic nature of steroid hormones, liquid-liquid extraction (LLE) with
non-polar solvents (e.g., MTBE, EtAc and dichloromethane) and SPE with reversed-phase
sorbents (e.g., C18) were considered appropriate to efficiently extract the analytes from
urine and serum/plasma [19,20,26]. We compared the performance of LLE using
MTBE/EtAc (5:1, v/v) [20] and different types of SPE cartridges including Bond Elut C18,
Toxics 2022, 10, 687 8 of 15

3.2. Optimization of Sample Purification

Given the lipophilic nature of steroid hormones, liquid-liquid extraction (LLE) with
non-polar solvents (e.g., MTBE, EtAc and dichloromethane) and SPE with reversed-phase
sorbents (e.g., C18) were considered appropriate to efficiently extract the analytes from urine
and serum/plasma [19,20,26]. We compared the performance of LLE using MTBE/EtAc
(5:1, v/v) [20] and different types of SPE cartridges including Bond Elut C18, Bond Elut
Plexa, and Bond Elut NEXUS (Supplementary Material). The recoveries of most of the
target analytes through the extraction (LLE) and purification (SPE) steps were acceptable
(Table S2). However, strong matrix effect following SPE (Figure S2) resulted in poor resolution
of pregnenolone and 13 C2 -D2 -pregnenolone. Resolution of this compound was excellent in
serum samples after LLE. Therefore, samples were analysed using LC-MS/MS after LLE
with MTBE/EtAc (5:1, v/v).
In human urine, steroid hormones are present almost exclusively as phase II metabo-
lites. Thus, deconjugation is an essential step to measure the total concentrations of steroid
hormones. ALS enzyme was reported to be efficient for the deconjugation of phase II
metabolites of various endogenous and exogenous compounds [20,27]. Therefore, urine
samples were incubated with ALS enzyme prior to extraction.

3.3. Method Validation

A 16-point calibration curve was prepared at concentrations ranging from 0.05 to
2000 ng/mL, with 10–50 ng/mL of isotopically labelled internal standards. Calibration
curves prepared in pure solvent yielded R-values in the range of 0.9973–0.9999 for all
steroid hormones (Table S3). Matrix-matched calibration curves at fortification levels of
0.05–2000 ng/mL also exhibited excellent R-values in both synthetic urine (0.9974–0.9998)
and pooled human serum (0.9958–0.9998) (Tables 2 and 3).
Accuracy of the method was assessed by spike-recovery tests conducted in triplicate
in pooled human urine and pooled human serum. Average recoveries of each analyte at
different fortification levels (10, 20, and 200 ng/mL) were measured. In human urine, the
recoveries of all hormones (except for progesterone) at three fortification levels were in
the range of 81.7–117%, 85.0–120%, and 82.7–119%, respectively, and the relative standard
deviation (SD) of triplicate measurements was 1.00–15.5%, 0–13.0%, and 0.30–11.0%, respec-
tively. These results suggest acceptable accuracies for most of the analytes. Nevertheless,
recoveries of progesterone were in the range of 117–141% (Table 2). This may indicate that
certain untargeted hormone metabolites (or precursors) exhibit the same retention and
MRM transition with progesterone, considering their similar chemical structures [1]. In
human serum, the recoveries of all hormones at three fortification levels were in the range
of 88.5–119%, 80.0–118%, and 88.7–119%, respectively, and the SD of triplicate analyses was
in the range of 1.2–14.1%, 1–17.3%, and 1.0–8.8%, respectively (Table 3). The determined
accuracies of all analytes were similar to those reported in an earlier study (84–122%) [20].
Toxics 2022, 10, 687 9 of 15

Table 2. Method validation parameters for the analysis of 19 steroid hormones in human urine.

10 ng/mL 20 ng/mL 200 ng/mL

LOD LOQ
Analytes Ra (ng/mL) (ng/mL) ME% CV% CV% CV%
Recovery% Recovery% Recovery%
Intra-Day Inter-Day Intra-Day Inter-Day Intra-Day Inter-Day
Estrogen
Estrone 0.9990 0.17 0.56 −6.56 111 ± 9 8.39 3.24 114 ± 3 2.78 2.39 105 ± 5 4.61 0.55
Estradiol 0.9992 0.04 0.14 0.92 93.3 ± 11.5 12.4 2.84 120 ± 10 8.67 3.10 118 ± 11 9.05 4.08
Estriol 0.9981 0.13 0.42 −2.16 110 ± 5 4.18 9.68 109 ± 3 3.12 6.83 99.6 ± 5.3 5.31 3.65
Androgen
Testosterone 0.9997 0.14 0.46 −0.81 113 ± 5 4.20 0.71 119 ± 1 0.60 1.34 115 ± 3 2.10 1.71
5α-Dihydrotestosterone 0.9974 0.15 0.49 −2.51 82.5 ± 1.3 1.50 4.17 85.0 ± 1.9 2.20 3.21 82.7 ± 2.0 2.40 0.74
Androstenedione 0.9994 0.22 0.73 7.30 109 ± 13 11.7 2.39 109 ± 11 10.4 2.17 119 ± 7 6.20 3.68
Androstenediol 0.9994 0.23 0.76 0.49 102 ± 12 11.7 2.56 119 ± 8 6.40 0.56 99.8 ± 4.8 4.83 0.65
DEHA 0.9996 0.24 0.81 −1.67 117 ± 6 4.95 3.88 91.7 ± 0 0.00 4.27 105 ± 8 7.45 2.58
Progestogen
Progesterone 0.9990 0.15 0.50 −2.65 141 ± 11 7.50 3.93 132 ± 8 6.30 4.45 117 ± 8 6.60 4.77
Pregnenolone 0.9998 0.23 0.75 −2.48 106 ± 6 6.00 1.77 111 ± 1 0.90 2.70 110 ± 4 3.72 2.29
17α-OH-Progesterone 0.9997 0.21 0.70 0.75 110 ± 1 0.60 1.49 115 ± 13 11.2 2.31 113 ± 4 3.30 1.69
17α-OH-Pregnenolone 0.9998 0.12 0.41 −2.45 112 ± 4 3.10 3.86 112 ± 1 1.10 1.00 96.8 ± 4.0 4.20 2.43
Corticosteroid
Cortisone 0.9985 0.13 0.44 29.6 101 ± 12 11.5 4.53 115 ± 8 7.08 3.41 117 ± 4 3.47 4.25
Cortisol 0.9998 0.20 0.65 −1.74 81.7 ± 15.5 19.0 1.34 100 ± 9 9.35 1.20 96.5 ± 4.3 4.49 3.12
11-Deoxycortisol 0.9995 0.19 0.64 5.84 112 ± 3 2.37 2.63 116 ± 4 3.80 4.37 106 ± 3 2.76 1.17
11-Deoxycorticosterone 0.9998 0.25 0.83 −2.47 97.1 ± 9.9 10.2 1.29 99.2 ± 1.4 1.45 2.42 99.9 ± 2.0 2.02 1.58
11-Dehydrocorticosterone 0.9990 0.28 0.92 5.41 86.7 ± 5.5 6.35 2.56 90.2 ± 3.2 3.56 1.82 84.1 ± 2.5 2.93 3.20
Corticosterone 0.9993 0.26 0.85 5.52 107 ± 4 3.37 2.20 101 ± 2 1.72 1.39 101 ± 1.3 1.31 1.13
Aldosterone 0.9997 0.19 0.63 4.42 95.5 ± 2.6 2.77 7.16 101 ± 10 9.47 7.59 90.0 ± 0.3 0.32 4.59
Abbreviation: DEHA, dehydroepiandrosterone; ME, matrix effect; LOD, limit of detection; LOQ, limit of quantification; CV, coefficient of variation. a R value in urine matrix.
Toxics 2022, 10, 687 10 of 15

Table 3. Method validation parameters for the analysis of 19 steroid hormones in human serum.

10 ng/mL 20 ng/mL 200 ng/mL

LOD LOQ
Analytes Ra (ng/mL) (ng/mL) ME% CV% CV% CV%
Recovery% Recovery% Recovery%
Intra-Day Inter-Day Intra-Day Inter-Day Intra-Day Inter-Day
Estrogen
Estrone 0.9998 0.11 0.38 1.09 112 ± 5 4.08 2.18 103 ± 3 2.97 6.06 97.3 ± 3.7 3.79 0.78
Estradiol 0.9989 0.14 0.48 0.92 88.5 ± 3.8 4.33 3.17 92.3 ± 8.9 9.63 1.14 93.3 ± 1.8 3.29 2.68
Estriol 0.9982 0.17 0.55 −6.49 110 ± 2 1.89 2.46 114 ± 5 3.97 2.39 99.0 ± 3.1 3.15 1.13
Androgen
Testosterone 0.9958 0.22 0.75 −0.12 118 ± 8 6.37 2.16 116 ± 6 5.19 1.26 119 ± 1 0.64 2.63
5α-Dihydrotestosterone 0.9998 0.18 0.60 0.00 104 ± 7 6.28 4.78 114 ± 3 2.67 0.78 108 ± 1 0.80 3.02
Androstenedione 0.9985 0.16 0.52 18.2 114 ± 3 2.68 4.09 117 ± 3 2.58 1.48 110 ± 3 2.73 2.08
Androstenediol 0.9985 0.24 0.81 5.34 96.1 ± 1.2 1.20 3.88 104 ± 6 5.30 3.00 99.7 ± 1.4 1.45 2.20
DEHA 0.9990 0.17 0.56 −10.2 102 ± 5 5.05 6.43 102 ± 8 7.62 2.40 107 ± 3 2.84 3.89
Progestogen
Progesterone 0.9997 0.17 0.58 −7.08 110 ± 5 4.48 4.40 115 ± 5 4.24 6.72 110 ± 3 2.62 5.82
Pregnenolone 0.9992 0.35 1.18 −0.62 96.6 ± 12.1 12.5 11.5 91.5 ± 1.3 1.45 8.55 100 ± 2 2.18 1.75
17α-OH-Progesterone 0.9994 0.13 0.42 2.99 109 ± 6 5.36 2.95 108 ± 2 1.84 1.42 100 ± 1 1.00 1.00
17α-OH-Pregnenolone 0.9974 0.24 0.80 −13.9 101 ± 3 3.04 5.80 100 ± 7 6.60 4.14 92.8 ± 3.5 3.73 2.51
Corticosteroid
Cortisone 0.9990 0.25 0.84 38.7 119 ± 9 7.33 4.33 118 ± 8 6.58 1.04 119 ± 5 4.21 2.61
Cortisol 0.9981 0.24 0.79 −3.48 90.0 ± 14.1 15.7 1.24 80.0 ± 17.3 21.7 0.96 94.0 ± 8.8 9.32 2.06
11-Deoxycortisol 0.9997 0.17 0.58 7.22 107 ± 3 2.46 2.55 111 ± 2 1.88 1.99 105 ± 3 2.72 1.91
11-Deoxycorticosterone 0.9989 0.21 0.70 0.64 97.2 ± 1.6 1.68 1.01 103 ± 4 3.67 2.22 96.0 ± 3.5 3.61 2.04
11-Dehydrocorticosterone 0.9981 0.23 0.78 6.76 106 ± 9 8.04 7.61 107 ± 1 1.08 4.73 103 ± 2 2.03 4.22
Corticosterone 0.9995 0.17 0.56 −11.0 94.7 ± 6.4 6.71 0.16 100 ± 4 4.36 2.66 88.7 ± 1.3 1.49 1.83
Aldosterone 0.9997 0.22 0.73 9.73 102 ± 6 5.59 1.41 106 ± 5 4.34 4.74 98.8 ± 4.7 4.70 1.11
Abbreviation: DEHA, dehydroepiandrosterone; ME, matrix effect; LOD, limit of detection; LOQ, limit of quantification; CV, coefficient of variation. a R value in serum matrix.
Toxics 2022, 10, 687 11 of 15

We further assessed and validated the accuracy of the method through the analysis
of NIST human plasma SRM1950. The concentrations of testosterone, progesterone, and
cortisol determined in our method agreed well with the certified reference values, with
variations within 11% (Table 4), further confirming validity of the developed method.

Table 4. Steroid hormones measured in plasma Standard Reference Material (SRM1950) using the
method developed in this study and compared to the certified reference values. Reference values
were only available for cortisol, progesterone, and testosterone.

Reference Value
Analytes Our Results (ng/mL) Variation%
(ng/mL)
Estrogen
Estrone 0.14 n.a. –
Estradiol <LOD n.a. –
Estriol <LOD n.a. –
Androgen
Testosterone 2.47 2.214 11
5α-Dihydrotestosterone 0.20 n.a. –
Androstenedione 0.74 n.a. –
Androstenediol 0.58 n.a. –
DEHA 1.99 n.a. –
Progestogen
Progesterone 1.36 1.482 8.6
Pregnenolone 0.41 n.a. –
17α-OH-Progesterone 0.58 n.a. –
17α-OH-Pregnenolone 0.94 n.a. –
Corticosteroid
Cortisol 81.4 83.9 3
Cortisone 20.9 n.a. –
11-Deoxycortisol 0.17 n.a. –
11-Deoxycorticosterone <LOD n.a. –
11-Dehydrocorticosterone 0.67 n.a. –
Corticosterone 1.57 n.a. –
Aldosterone <LOD n.a. –
Abbreviation: n.a., not available; DEHA, dehydroepiandrosterone; LOD, limit of detection.

We assessed precision of the method as intra-day and inter-day variations from repeated
analysis of fortified human urine and serum samples. In human urine, the intra-day CVs
of target analytes fortified at 10, 20, and 200 ng/mL were 0.60–19.0%, 0–11.2%, and 0.32–
9.05%, respectively, whereas the inter-day CVs were 0.71–9.68%, 0.56–7.59%, and 0.55–4.77%,
respectively (Table 2). In human serum, the intra-day CVs were 1.20–15.7%, 1.08–21.7%,
and 0.64–9.32%, respectively, whereas the inter-day CVs were 0.16–11.5%, 0.78–8.55%, and
0.78–5.82%, respectively (Table 3). These results suggested acceptable precision (CVs < 20%)
for all analytes except cortisol in serum (9.32–21.7%). The relatively low precision of cortisol
was likely due to its high background concentration in urine and serum (Tables 4 and S4).
The method precision calculated in this study was similar to that reported in an earlier
study (intra-day CV: 2–11%; inter-day CV: 5–24%) [20].
Matrix effect is a common phenomenon in ESI-LC-MS analysis, produced by ionization
enhancement or suppression by matrix components. Typically, values in the range of
−20 to +20% are considered as weak suppression/enhancement. Use of isotopically
labelled internal standards of analytes (and isotopic dilution method of quantification)
is an acceptable practice to compensate for matrix effects. In this study, all analytes
except cortisone exhibited weak matrix effects in both urine (−6.56 to 7.30%) and serum
(−13.9 to 18.2%), indicating that the optimized method adequately removed interfering
matrix components (Tables 2 and 3). However, a moderate ion enhancement was found
for cortisone in urine (29.6%) and serum (38.7%), which could be explained by the lack of
isotope labelled cortisone available in this study (13 C3 -cortisol was used as the internal
standard for cortisone). Use of isotopically labelled cortisone would reduce the uncertainty
in measurement associated with matrix-effect for this analyte.
The sensitivity of the method was determined as LOD and LOQ in both human urine
and serum. In human urine, the LODs ranged from 0.04 ng/mL (estradiol) to 0.28 ng/mL (11-
 in urine (29.6%) and serum (38.7%), which could be explained by the lack of isotope la-
belled cortisone available in this study (13C3-cortisol was used as the internal standard for
cortisone). Use of isotopically labelled cortisone would reduce the uncertainty in meas-
urement associated with matrix-effect for this analyte.
The sensitivity of the method was determined as LOD and LOQ in both human urine
Toxics 2022, 10, 687 12 of 15
and serum. In human urine, the LODs ranged from 0.04 ng/mL (estradiol) to 0.28 ng/mL
(11-dehydrocorticosterone), and the LOQs were between 0.14 ng/mL (estradiol) and 0.92
ng/mL (11-dehydrocorticosterone) (Table 2). In human serum, the LODs ranged from 0.11
dehydrocorticosterone),
ng/mL (estrone) to 0.35and the LOQs
ng/mL were between
(pregnenolone), and0.14
theng/mL (estradiol)
LOQs were and 0.92
between 0.38 ng/mL
ng/mL
(11-dehydrocorticosterone)
(estrone) and 1.18 ng/mL (pregnenolone) (Table 3). Thus, the method providesng/mL
(Table 2). In human serum, the LODs ranged from 0.11 adequate(es-
trone) to 0.35 ng/mL (pregnenolone), and the LOQs were between 0.38 ng/mL
sensitivity for the determination of 19 steroid hormones in human urine and se- (estrone) and
1.18 ng/mL (pregnenolone) (Table 3). Thus, the method provides adequate sensitivity for
rum/plasma. The method sensitivities were similar to those reported in the literature (e.g.,
the determination of 19 steroid hormones in human urine and serum/plasma. The method
LODs: 0.20–1.00 ng/mL in urine [20], and 0.04–1.35 in fetal bovine serum [5]) although the
sensitivities were similar to those reported in the literature (e.g., LODs: 0.20–1.00 ng/mL
earlier studies used different methods to calculate LOD. In particular, the LODs for estro-
in urine [20], and 0.04–1.35 in fetal bovine serum [5]) although the earlier studies used
gens in urine in our study (0.04–0.17 ng/mL) were significantly lower than those reported
different methods to calculate LOD. In particular, the LODs for estrogens in urine in our
in an earlier study (0.20–0.83 ng/mL) [20].
study (0.04–0.17 ng/mL) were significantly lower than those reported in an earlier study
(0.20–0.83 ng/mL) [20].
3.4. Method Application
The validated
3.4. Method method was applied for the analysis of 20 spot urine samples collected
Application
fromThehealthy volunteers
validated method (11was
males aged 18–64
applied for they,analysis
and 9 females aged
of 20 spot 15–59
urine y) during
samples May–
collected
June 2022 from New York City. Sixteen analytes were found with detection
from healthy volunteers (11 males aged 18–64 y, and 9 females aged 15–59 y) during May– frequencies
(DFs)2022
June ≥ 80% in urine
from Newsamples.
York City.The average
Sixteen concentrations
analytes were foundof allwith
analytes ranged
detection from 0.34
frequencies
to 284≥ng/mL.
(DFs) 80% inIn bothsamples.
urine males and
Thefemales,
averageDEHA was the most
concentrations abundantranged
of all analytes (accounting for
from 0.34
39%
to 284and 32% of
ng/mL. Inthe total
both concentrations
males and females,inDEHA
males was
and the
females,
most respectively), followed for
abundant (accounting by
estradiol (20% and 23%, respectively), cortisol (13% and 14%, respectively),
39% and 32% of the total concentrations in males and females, respectively), followed by cortisone (8%
and 9%, respectively),
estradiol (20% and 23%,and androstenediol
respectively), (8%(13%
cortisol and 9%,
and respectively) (Figure
14%, respectively), 4 and Table
cortisone (8%
S4). 9%,
and Representative
respectively), chromatograms of all(8%
and androstenediol analytes
and 9%,in respectively)
urine samples are shown
(Figure 4 and in Figure
Table S4).
S3.
Representative chromatograms of all analytes in urine samples are shown in Figure S3.

Figure 4.
Figure 4. Concentrations
Concentrations (a)
(a) and
and relative
relative distribution
distribution (b)
(b) of
of 19
19 steroid
steroid hormones
hormones measured
measured in
in urine
urine
from 11 males and 9 females from New York, United States. Mean concentrations were used in the
from 11 males and 9 females from New York, United States. Mean concentrations were used in the
calculation of the percent contribution.
calculation of the percent contribution.

The method was applied in the analysis of human plasma by measuring all analytes in
SRM1950. Among the 19 steroid hormones, 15 analytes were quantified at concentrations
ranging from 0.14 (estrone) and 81.4 ng/mL (cortisol). Aldosterone, 11-deoxycorticosterone,
estradiol, and estriol were below the respective LODs in the sample. In pooled human
serum purchased from Sigma-Aldrich, 12 out of the 19 analytes were found at concentra-
tions in the range of 0.23 (11-deoxycortisol) to 72.5 ng/mL (cortisol) (Table S5). Represen-
tative chromatograms of all analytes in serum samples are shown in Figure S4. Overall,
Toxics 2022, 10, 687 13 of 15

the method is suitable for the determination of 19 steroid hormones in human urine and
serum/plasma of the general population.

4. Conclusions
We developed and validated a HPLC-MS/MS method for simultaneous determination
of 19 steroid hormones in human urine and serum/plasma. Liquid-liquid extraction with
MTBE/EtAc (5:1, v/v) yielded acceptable recoveries of all analytes from these matrices.
Chromatographic and mass spectrometric conditions were optimized to separate and detect
the hormones at physiologically relevant concentrations. Dansylation of estrone, estradiol,
and estriol improved their chromatographic resolution and sensitivities.
The method was validated for accuracy, precision, sensitivity, linearity, and through
the analysis of certified plasma standard reference material. The method was applied in the
analysis of 19 hormones in real human urine and serum/plasma. The method can also be
used in the analysis of steroid hormones in other matrices (e.g., hair and saliva) with slight
modifications. The method can be applied in the analysis of steroid hormones in human
urine and serum/plasma in population-based biomonitoring studies.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/toxics10110687/s1. Supplementary method: sample cleanup
procedure using SPE. Figure S1. Representative LC-MS/MS chromatograms of 19 steroid hormones
without derivatization. Concentrations of the target analytes were 100 ng/mL; Injection volume was
10 µL. Figure S2. Comparison of pregnenolone in serum following extraction with LLE, Bond Elut
C18, Bond Elut Plexa, and Bond Elut NEXUS. Analyte concentration was 100 ng/mL, and the injection
volume was 10 µL. Figure S3. Representative chromatograms of 19 steroid hormones found in human
urine (injection volume: 10 µL). Figure S4. Representative chromatograms of 19 steroid hormones
found in human serum (injection volume: 10 µL). Table S1. Optimized MRM parameters for the
estrogens. Table S2. Comparison of the spike-recoveries of all analytes in serum following extraction
with LLE, Bond Elut C18 (60 mg/3 mL), Bond Elut Plexa (60 mg/3 mL), and Bond Elut NEXUS
(60 mg/3 mL). Analytes were spiked at 100 ng/mL, injection volume was 10 µL. Table S3. R value
of the calibration curve of all analytes in neat solution. Table S4. Steroid hormone concentrations
measured in twenty human urine samples from the general population in New York, USA, in 2022.
Table S5. Steroid hormone concentrations measured in the pooled human serum purchased from
Sigma-Aldrich.
Author Contributions: Z.-M.L.: Methodology, Data curation, Formal analysis, Writing—original
draft. K.K.: Conceptualization, Funding acquisition, Supervision, Writing—review and editing. All
authors have read and agreed to the published version of the manuscript.
Funding: The research reported here was supported, in part, by the US National Institute of Environ-
mental Health Sciences (NIEHS) under award number U2CES026542 (KK). The content is solely the
responsibility of the authors and does not necessarily represent the official views of the NIEHS.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Institutional Review Board of the New York University for the
analysis of de-identified urine samples (under exempt category) to demonstrate application of the
method developed in this study.
Informed Consent Statement: Informed consent was waived as the study was deemed exempt
human study and only de-identified specimens were analyzed.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare that there are no conflict of interest.
Toxics 2022, 10, 687 14 of 15

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