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\begin{document}

\title{Profiling of urinary steroids by lithium ion adduction-based UPLC-MS/MS}



\author[1]{Toshifumi Takao}%
\author[1]{Yue Pan}%
\author[1]{Qiuyi Wang}%
\author[1]{Mengyao Chen}%
\affil[1]{Osaka Daigaku Tanpakushitsu Kenkyujo}%


\vspace{-1em}



  
  \date{June 04, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
\textbf{RATIONALE:} Urine contains free and conjugated steroids. Total
and free steroids were comprehensively quantified; UPLC-MS/MS based on
Li adduction allowed for detecting thirteen 3-OH-containing steroids,
two of which were detected in human urine for the first time.
\textbf{METHODS:} Free urinary steroids were isolated by solid-phase
extraction (SPE) with 80\% acetonitrile. The total steroids were
prepared by enzymatic treatment of urine with a cocktail of sulfatase
and glucronidase, protein precipitation, and separation with the above
SPE. The free and total steroids were separately analyzed by UPLC-MS/MS
with and without introduction of Li \textsuperscript{+} solution. The
steroids were quantified by two standard curves created using product
ion transitions derived from MH \textsuperscript{+} and {[}M+Li{]}
\textsuperscript{+}. \textbf{RESULTS:} Two groups of human urine, male
and female urine, were analyzed. The absolute amount of each steroid was
determined based on creatinine levels. The differences between the male
and female groups are clearly attributable to sex steroids. 7-OH P5 and
7-OH DHEA were, for the first time, quantified in the total steroids of
female urine, and the latter was identified in both female and male
urine. \textbf{CONCLUSIONS:} By combining UPLC-MS/MS based on lithium
ion incorporation with conventional UPLC-MS/MS, a total of 29 steroids
were identified in human urine containing two newly found steroids.%
\end{abstract}%



\sloppy


\textbf{Profiling of urinary steroids bylithium ion adduction}
\textbf{-based UPLC-MS/MS}

Yue Pan\textsuperscript{1}, Qiuyi Wang\textsuperscript{1+}, Mengyao
Chen\textsuperscript{1}, and Toshifumi Takao\textsuperscript{1,*}

\textsuperscript{1} Institute for Protein Research, Osaka University,
Osaka 565-0871, Japan

* To whom correspondence should be addressed

Prof. Toshifumi TAKAO

Laboratory of Protein Profiling and Functional Proteomics, Institute for
Protein Research, Osaka University, 3-2 Yamadaoka, Suita-shi, Osaka
565-0871, Japan; TEL: 06-6879-4312; FAX: 06-6879-4332; E-mail:
tak@protein.osaka-u.ac.jp

\textsuperscript{+}Present address: Shimadzu Corporation, Kyoto
604-8511, Japan

\textbf{Abbreviations} : 3-OH steroids, Steroids that contain a
3-hydroxyl group; 11-OH-An, 11-\selectlanguage{greek}β-\selectlanguage{english}hydroxyandrosterone; 16-OH-E1,
16-hydroxyestrone; 6-OH-E1-\textsuperscript{13}C\textsubscript{3},
16-hydroxyestrone-2,3,4-\textsuperscript{13}C\textsubscript{3};
17-OH-P4, 17\selectlanguage{greek}α-\selectlanguage{english}hydroxyprogesterone;
17-OH-P4-\textsuperscript{13}C\textsubscript{3,}17\selectlanguage{greek}α-\selectlanguage{english}hydroxyprogesterone-2,3,4-\textsuperscript{13}C\textsubscript{3};
17-OH-P5, 17\selectlanguage{greek}α-\selectlanguage{english}hydroxypregnenolone; 17-OH P5-d\textsubscript{3},
17\selectlanguage{greek}α-\selectlanguage{english}hydroxypregnenolone-21,21,21-d\textsubscript{3}; 7-OH DHEA,
7\selectlanguage{greek}α-\selectlanguage{english}hydroxydehydroepiandrosterone; 7-OH P5, 7\selectlanguage{greek}α-\selectlanguage{english}hydroxypregnenolone; ACN,
acetonitrile; AcOH, acetate acid; AE, androstenedione;
AE-\textsuperscript{13}C\textsubscript{3},
androstene-3,17-dione-2,3,4-\textsuperscript{13}C\textsubscript{3};
al-Preg, allopregnenolone; An, androsterone; APD, alphadolone; AT,
adrenosterone; CID, collision induced dissociation; COB, corticosterone;
COB-d\textsubscript{4}, corticosterone-9,11,11,12-d\textsubscript{4};
COL, Cortisol; ~COL-d\textsubscript{4},
cortisol-9,11,12,12-d\textsubscript{4}; COR, cortisone;
COR-\textsuperscript{13}C\textsubscript{3},
cortisone-2,3,4-\textsuperscript{13}C\textsubscript{3}; COS,
11-deoxycortisol; DHEA, dehydroepiandrosterone; DHEA-d\textsubscript{5},
dehydroepiandrosterone-2,2,3,4,4-d\textsubscript{5}; DHT,
dehydrotestosterone; DOC, 11-deoxycorticosterone; E1, estrone;
E1-\textsuperscript{13}C\textsubscript{3},
estrone-2,3,4-\textsuperscript{13}C\textsubscript{3}; E2, estradiol; E3,
estriol; E3-d\textsubscript{3}, estriol-d\textsubscript{3}; ESI,
electrospray ionization; LiCl, lithium chloride; MeOH, methanol; MRM
multiple reaction monitoring, P4, progesterone;
P4-\textsuperscript{13}C\textsubscript{3},
progesterone-2,3,4-\textsuperscript{13}C\textsubscript{3}; P5,
pregnenolone;
P5-\textsuperscript{13}C\textsubscript{2},d\textsubscript{2},
pregnenolone-\textsuperscript{13}C\textsubscript{2},d\textsubscript{2};
SPE, solid-phase-extraction; TE, testosterone;
TE-\textsuperscript{13}C\textsubscript{3,}testosterone-2,3,4-\textsuperscript{13}C\textsubscript{3;}
THB, tetrahydrocorticosterone; TH-COL, tetrahydrocortisol; TH-COR,
tetrahydrocortisone; TH-COR-d\textsubscript{6},
tetrahydrocortisone-2,2,4,4,21,21-d\textsubscript{6}-d\textsubscript{6};
TH-DOC, 3\selectlanguage{greek}β,\selectlanguage{english}5\selectlanguage{greek}α-\selectlanguage{english}tetrahydrodeoxycorticosterone; THS,
tetrahydrodeoxycortisol.

Running title: Steroid profiling of human urine

\textbf{Abstract} :

\textbf{RATIONALE:} Urine contains free and conjugated steroids. Total
and free steroids were comprehensively quantified; UPLC-MS/MS based on
Li adduction allowed for detecting thirteen 3-OH-containing steroids,
two of which were detected in human urine for the first time.

\textbf{METHODS:} Free urinary steroids were isolated by solid-phase
extraction (SPE) with 80\% acetonitrile. The total steroids were
prepared by enzymatic treatment of urine with a cocktail of sulfatase
and glucronidase, protein precipitation, and separation with the above
SPE. The free and total steroids were separately analyzed by UPLC-MS/MS
with and without introduction of Li\textsuperscript{+} solution. The
steroids were quantified by two standard curves created using product
ion transitions derived from MH\textsuperscript{+} and
{[}M+Li{]}\textsuperscript{+}.

\textbf{RESULTS:} Two groups of human urine, male and female urine, were
analyzed. The absolute amount of each steroid was determined based on
creatinine levels. The differences between the male and female groups
are clearly attributable to sex steroids. 7-OH P5 and 7-OH DHEA were,
for the first time, quantified in the total steroids of female urine,
and the latter was identified in both female and male urine.

\textbf{CONCLUSIONS:} By combining UPLC-MS/MS based on lithium ion
incorporation with conventional UPLC-MS/MS, a total of 29 steroids were
identified in human urine containing two newly found steroids.

\textbf{Keywords:} human urine, conjugated steroids, Li adduction,
UPLC-MS/MS

\textbf{Introduction}

Steroids play important roles in various physiological functions of the
organism, including brain development, behavior, cognition,
neuroplasticity, and neuroinflammation, and are metabolized and excreted
in the urine as conjugated forms\textsuperscript{1-4}. Transient
concentrations of each steroid in the blood indicate a physiological
state, but these concentrations change rapidly in response to various
physiological and even psychological changes\textsuperscript{5,6}.

Urinary steroids represent the total amount of each steroid circulating
in the blood over a given time period. Profiling of urinary steroids
should be important for diagnosing relatively long-term physiological
changes such as chronic disease, breast cancer and prostate
cancer\textsuperscript{1,7,8}. Steroid hormones are enzymatically
synthesized from cholesterol and classified into various types of
steroids, including estrogens (female reproductive steroids), androgens
(male reproductive steroids), progestogens (pregnancy steroids), and
corticosteroids (stress steroids)\textsuperscript{2,4}. After
circulating in the blood and functioning in their respective organs,
these steroids are enzymatically converted to inactive substances linked
with sulfate or glucuronic acid in several organs such as liver and
prostate, and excreted in urine\textsuperscript{4,7,9}. Due to their
diverse structures, these conjugated steroids are usually converted back
to free steroids for the analysis; The advantage of urine is that it is
non-invasive, therefore it has no pain and is easy to collect in large
amounts\textsuperscript{10}. LC/ESI-MS/MS has become a popular method
for steroid analysis in biological samples due to its high sensitivity,
high selectivity and the ability to run multiple analytes
simultaneously\textsuperscript{11-13}. However, 26 of the 29 steroids
contain hydroxyl groups in their backbone, which are prone to
dehydration when ionized by positive-ion ESI. Such dehydration
diversifies the precursor ion, thereby reducing sensitivity and
hindering their quantification by MRM methods. To overcome this issue,
we recently reported a method to protect steroid molecules from
dehydration by using Li+ as the adduct ion to steroid (``Li
method'')\textsuperscript{14}.

Application of the Li method to the analysis of human urine resulted in
the detection of 13 3-OH steroids with 1.3-20 times greater sensitivity
than conventional LC/ESI-MS/MS (``H method''). Other steroids (16
species) were identified by the H method. As a result, a total of 29
steroids were identified.

\textbf{Materials and Methods:}

\textbf{Chemicals}

HPLC grade methanol, acetonitrile, 99.998\% trace metals basis lithium
chloride, analytical-reagent-grade acetate acid, L-Ascorbic acid,
\selectlanguage{greek}β-\selectlanguage{english}glucuronidase-arylsulfatase from Helix pomatia, sodium bicarbonate,
corticosterone, cortisol, cortisone, dehydroepiandrosterone,
dehydrotestosterone , 11-deoxycorticosterone, 11-deoxycortisol, estriol,
17\selectlanguage{greek}α-\selectlanguage{english}hydroxypregnenolone, 17\selectlanguage{greek}α-\selectlanguage{english}hydroxyprogesterone, pregnenolone,
progesterone, testosterone,
androstene-3,17-dione-2,3,4-\textsuperscript{13}C\textsubscript{3},
corticosterone-9,11,11,12-d\textsubscript{4},
cortisol-9,11,12,12-d\textsubscript{4},
cortisone-2,3,4-\textsuperscript{13}C\textsubscript{3},
dehydroepiandrosterone-2,2,3,4,4-d\textsubscript{5},
estrone-2,3,4-\textsuperscript{13}C\textsubscript{3},
17\selectlanguage{greek}α-\selectlanguage{english}hydroxypregnenolone-21,21,21-d\textsubscript{3},
17\selectlanguage{greek}α-\selectlanguage{english}hydroxyprogesterone-2,3,4-\textsuperscript{13}C\textsubscript{3},
pregnenolone-20,21-\textsuperscript{13}C\textsubscript{2}-16,16-d\textsubscript{2}solution,
progesterone-2,3,4-\textsuperscript{13}C\textsubscript{3} and
testosterone-2,3,4-\textsuperscript{13}C\textsubscript{3} were purchased
from Sigma-Aldrich (Tokyo, Japan). Alphadolone , allopregnenolone ,
tetrahydrocortisol, tetrahydrocortisone, 11-\selectlanguage{greek}β-\selectlanguage{english}hydroxyandrosterone,
tetrahydrocorticosterone, tetrahydrodeoxycorticosterone,
tetrahydrodeoxycortisol, 16-hydroxyestrone,
16-hydroxyestrone-2,3,4-\textsuperscript{13}C\textsubscript{3},
estriol-d\textsubscript{3} and
tetrahydrocortisone-d\textsubscript{6}were supplied by Toronto Research
Chemicals (North York, Canada). Adrenosterone, androsterone,
androstenedione, estrone, and estradiol was purchased from Tokyo
Chemical Industry (Tokyo, Japan). 7\selectlanguage{greek}α-\selectlanguage{english}hydroxydehydroepiandrosterone and
7\selectlanguage{greek}α-\selectlanguage{english}hydroxypregnenolone were supplied by Nacalai tesque (Kyoto, Japan).
HPLC grade formic acid was obtained from FUJIFILM Wako Pure Chemical Co.
(Osaka, Japan). Ultrapure water was prepared using a Organo puric \selectlanguage{greek}ω
\selectlanguage{english}(Tokyo, Japan). The HF Bond Elute C18 (1 mL, 50 mg) was purchased from
Agilent Technologies, Inc. (Santa Clara, CA). Creatinine (urinary)
Colorimetric Assay kit was purchased from Cayman Chemical (Ann Arbor,
MI). The amount of creatinine (mg) was measured according to the
manufacturer's manual.

\textbf{Human urine and ethical approval}

The healthy samples of human urine (3 males, 3 females) without
personally identifiable information were purchased from Innovative
Research, Inc. (Novi, MI). This study was approved by the Ethics
Committee of the Institute for Protein Research, Osaka University (No.
2020-3). Human studies abided by the Declaration of Helsinki principles.

\textbf{Sample preparation}

Steroid extraction strategy should be developed, taking into
consideration nature of the matrix, cleaning and pre-concentration step,
and detection of conjugated steroid\textsuperscript{1,15,16}. 240 \selectlanguage{greek}µ\selectlanguage{english}L
urine were first mixed with the internal standard mixture solution (2.4
uL) (see \textbf{EXPERIMENTAL SECTION} in the ``\textbf{Supplemental
information} ''), mixed with 240 \selectlanguage{greek}µ\selectlanguage{english}L of freshly prepared 0.15 M sodium
acetate buffer (pH 4.6) containing 1.5 mg \emph{L} -ascorbic acid and 4
\selectlanguage{greek}µ\selectlanguage{english}L of \selectlanguage{greek}β-\selectlanguage{english}glucuronidase-arylsulfatase solution. After the reaction, the
solution was allowed to stand at 37 \selectlanguage{ngerman}°C for 16 h, 960 \selectlanguage{greek}µ\selectlanguage{english}L of ACN was mixed
in a 2 mL Eppendorf tube (Eppendorf AG, Hamburg, Germany), vortexed for
30 s and allowed to stand at 4 \selectlanguage{ngerman}°C for 30 min. The mixture was
centrifuged at 21,000 x g for 15 min at 4°C and the protein precipitate
was spun down. The supernatant was transferred to a 15 mL Eppendorf tube
and 8.16 mL of H\textsubscript{2}O was added to give a final
concentration of 10\% ACN (v/v). The mixture was centrifuged at 19,000 x
g for 15 min at 4°C and the supernatant was loaded on a Bond Elute C18
column (50 mg) that had been washed with 1 mL of aqueous 80\% ACN twice
and equilibrated with 1 mL of aqueous 10\% ACN twice. Samples were
washed three times with 1 mL of 10\% ACN and collected with 1 mL of 80\%
ACN in 1.5 mL Eppendorf tubes. The eluate was evaporated to dryness
using a speed-vac and the resulting residue was redissolved in 24 \selectlanguage{greek}µ\selectlanguage{english}L of
40\% MeOH. The mixture was centrifuged at 20,000 x g for 15 min at room
temperature and the supernatant was transferred to a 250 \selectlanguage{greek}µ\selectlanguage{english}L inactivated
glass insert. The insert was placed in a vial and subjected to LC-MS/MS.

\textbf{LC/ESI-MS/MS}

The UPLC-MS/MS was performed according to the method described
previously\textsuperscript{14} using an Agilent 1290 Infinity II and
6470 triple quadrupole mass spectrometer equipped with an ESI ion source
(Agilent Technologies, Inc. Santa Clara, CA). Briefly, the samples were
separated with an Agilent Eclipse Plus C18 RRHD 2.1x100 mm, 1.8 \selectlanguage{greek}μ\selectlanguage{english}m
column by using solvent A (0.1\% formic acid in deionized water) and
solvent B (0.1\% formic acid in MeOH). The elution gradient was from
40-80.0 \% B from 0 to 8 min, maintained at 80 \% B from 8 to 10 min,
80-40.0 \% from 10.0 to 10.10 min and held at 40 \% B from 10.1 to 13.1
min. The injection volume was 20 \selectlanguage{greek}µ\selectlanguage{english}L. The MRM mode was applied for the
detection and quantitation of all steroids with two transitions
optimized for each targeted compound (Table S1). The post-column
addition of 0.2 mM LiCl in H\textsubscript{2}O was carried out with an
Agilent 1100 binary pump as the auxiliary pump. The column effluent (0.4
mL min\textsuperscript{-1}) and auxiliary solution (0.4 mL
min\textsuperscript{-1}) were mixed at the T-connector and passed
through two in-line filters (Agilent 1290 inline filter, 0.3\selectlanguage{greek}μ\selectlanguage{english}m), which
were connected in tandem, prior to reaching to the ion source (Figure
S1). The auxiliary pump was off when conducting the ``H method''.

\textbf{Data processing}

Data were acquired using an Agilent MassHunter Acquisition system, and
processed using Agilent MassHunter Quantitive Analysis, Microsoft Excel,
and OriginPro 2018J (Academic).

\textbf{Results and discussion}

Free and total steroids were quantified for male and female urine.
Steroids were comprehensively analyzed using the Li and H methods. As
discussed in the previous paper, 3-OH steroids severely undergo
dehydration when ionized in positive ion mode\textsuperscript{14}.
Furthermore, additional OH group(s) in the backbone, such as7-OH DHEA
and 7-OH P5, further promote dehydration during ionization and those are
difficult to detect by conventional LC/ESI-MS/MS. The adduction of Li
ions to steroid molecules in the ionization could protect them from
dehydration, resulting in an increase in ion intensity of the precursor
ion as a lithiated form and efficient generation of specific fragment
ions, especially, for 3-OH steroids\textsuperscript{14} (Figure 2 and
Figure S2). For 3-ketosteroids, the ''H method'' could be used because
they do not contain such hydroxyl groups. Furthermore, the analytical
settings of the H-method and the Li-method are very similar, so they can
be operated in a consecutive manner with a regular LC-MS/MS, allowing
for a more comprehensive analysis of multi-class steroids in
bio-samples. Figure 2 compares MRM chromatograms of free and total 7-OH
DHEA and 7-OH P5 from female and male specimens; 7-OH P5 was only
observed in total steroids of female specimen, while 7-OH DHEA was
observed in free and total steroids prepared from female and male
samples. These two steroids were first identified by the Li method in
human urine. When these steroids were measured by the H-method, 7-OH P5
was barely observed and 7-OH DHEA was likely to be observed as a
shouldered peak, but the retention time and peak height ratio of the two
transitions differed significantly from the standard one (Figure 2,
right panel). This could be partly attributed to instability of the
protonated ion, MH\textsuperscript{+} or
(M-H\textsubscript{2}O+H)\textsuperscript{+} ions, in the ion source.
The adduction of a Li ion to steroid in the gas phase should protect it
from the dehydration or degradation during ionization. Indeed, the
sensitization ratios of 7-OH-DHEA and 7-OH-P5 were 7.1 and 11.1,
respectively (Figure 2).

19 free and 29 total steroids were identified in the urine of men and
women. The amounts of each steroid was normalized by creatinine values
obtained separately for male and female samples (Table S5). The relative
composition of free steroids was different from the total steroids, the
majority of which consists of the conjugated forms. The predominant
urinary steroids (conjugated forms) were TH-COL, TH-COR, APH, COB, THB,
An, DHEA, and 11-OH-An. Female had relatively higher contents of
7-OH-P5, 16OH-E1, E3, P5, and 7-OH-DHEA than male, while male had higher
contents of DOC, AT, TH-DOC, TE, COR, COL THB, 11-OH-An and An, a result
that clearly reflects gender differences (Figure 3). Since 7-OH-P5,
16OH-E1, E3, P5, 7-OH-DHEA, AT, TE, 11-OH-An and An are sex steroids, it
is normal for significant differences between male and female urine
samples. The stress-related corticosteroids DOC, TH-DOC, COR, COL and
THB were common in male\textsuperscript{17}, however, this result needs
to be confirmed with more specimens. Note that the amounts of each
steroid were obtained for the mixture of three specimens of urine and
their levels were almost in the ranges of those reported
previously\textsuperscript{2,18-25} (Figure S5), which were obtained for
individuals' urine or 24-hours pooled urine\textsuperscript{22}, while
E2, THB were observed with higher levels than those reported previously.
It could be ascribable to inter-day or individual variation of steroid
metabolism.

The high sensitivity of the Li method also revealed the profile of free
steroids in urine (about 1.2\% of total steroid levels), although their
physiological function is unclear. It is worth mentioning that since the
method allowed the reliable quantification of 7-OH DHEA, which has been
reported to be involved in ``doping'', it will be useful for its
monitoring\textsuperscript{26}.

\textbf{Conclusions:}

Lithium ion adduction-based UPLC-MS/MS was applied to urinary steroid
analysis and thirteen 3-OH steroids were detected. Among them, 7-OH P5
and 7-OH DHEA were identified for the first time in human urine. A total
of 29 steroids and 19 free steroids were identified by combining
conventional methods to detect protonated ions. The urinary steroid
profile obtained in this study is useful for monitoring relatively
long-term physiological conditions and diagnosing chronic diseases.

\textbf{Figure 1.} Biosynthesis map of steroid hormones.

Urinary steroids identified in this study are shown, of which 3-OH
steroids are shown in green and newly identified steroids are shown in
red.

\textbf{Figure 2.} MRM chromatograms of newly identified 7-OH P5 (left)
and 7-OH DHEA (right) in human urine. Chromatograms were obtained for
standard (2 ng for each, topmost), female-total (second), male-total
(third), female-free (fourth), male-free (fifth) by the Li method (left)
and the H method (right). The values above the peaks indicate retention
times and the values in brackets indicate the peak-height ratio of the
two peaks in each chromatogram observed at the two MRM transitions set
for each compound (see Table S1). Typical fragmentation ions of
lithiated 3-OH steroids are depicted in their structures.

\textbf{Figure 3.} Pie chart of relative amounts of major steroids in
male (a) and female urine (b) and bar chart of free and total steroid
levels (ng/creatinine (mg)) obtained in male (top) and female (bottom)
urine (c). All the data were summarized in Table S5.

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\textbf{CRediT Classification:}

\textbf{YP} : Investigation, Conceptualization, Formal Analysis, Writing
-- Original Draft Preparation, Visualization,. \textbf{QW} :
Investigation, Conceptualization, Formal Analysis. \textbf{MC} :
Investigation, Review \& Editing. \textbf{TT} : Conceptualization,
Resources, Funding Acquisition, Supervision, Writing -- Review \&
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