Profiling
of urinary steroids bylithium ion
adduction -based
UPLC-MS/MS
Yue
Pan1, Qiuyi
Wang1†,
Mengyao Chen1, and Toshifumi
Takao1,*
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
†Present address: Shimadzu Corporation, Kyoto
604-8511, Japan
Abbreviations : 3-OH steroids, Steroids that contain a
3-hydroxyl group; 11-OH-An, 11-β-hydroxyandrosterone; 16-OH-E1,
16-hydroxyestrone; 6-OH-E1-13C3,
16-hydroxyestrone-2,3,4-13C3;
17-OH-P4, 17α-hydroxyprogesterone;
17-OH-P4-13C3,17α-hydroxyprogesterone-2,3,4-13C3;
17-OH-P5, 17α-hydroxypregnenolone; 17-OH P5-d3,
17α-hydroxypregnenolone-21,21,21-d3; 7-OH DHEA,
7α-hydroxydehydroepiandrosterone; 7-OH P5, 7α-hydroxypregnenolone; ACN,
acetonitrile; AcOH, acetate acid; AE, androstenedione;
AE-13C3,
androstene-3,17-dione-2,3,4-13C3;
al-Preg, allopregnenolone; An, androsterone; APD, alphadolone; AT,
adrenosterone; CID, collision induced dissociation; COB, corticosterone;
COB-d4, corticosterone-9,11,11,12-d4;
COL, Cortisol; COL-d4,
cortisol-9,11,12,12-d4; COR, cortisone;
COR-13C3,
cortisone-2,3,4-13C3; COS,
11-deoxycortisol; DHEA, dehydroepiandrosterone; DHEA-d5,
dehydroepiandrosterone-2,2,3,4,4-d5; DHT,
dehydrotestosterone; DOC, 11-deoxycorticosterone; E1, estrone;
E1-13C3,
estrone-2,3,4-13C3; E2, estradiol; E3,
estriol; E3-d3, estriol-d3; ESI,
electrospray ionization; LiCl, lithium chloride; MeOH, methanol; MRM
multiple reaction monitoring, P4, progesterone;
P4-13C3,
progesterone-2,3,4-13C3; P5,
pregnenolone;
P5-13C2,d2,
pregnenolone-13C2,d2;
SPE, solid-phase-extraction; TE, testosterone;
TE-13C3,testosterone-2,3,4-13C3; THB,
tetrahydrocorticosterone; TH-COL, tetrahydrocortisol; TH-COR,
tetrahydrocortisone; TH-COR-d6,
tetrahydrocortisone-2,2,4,4,21,21-d6-d6;
TH-DOC, 3β,5α-tetrahydrodeoxycorticosterone; THS,
tetrahydrodeoxycortisol.
Running title: Steroid profiling of human urine
Abstract :
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.
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+ solution. The
steroids were quantified by two standard curves created using product
ion transitions derived from MH+ and
[M+Li]+.
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.
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.
Keywords: human urine, conjugated steroids, Li adduction,
UPLC-MS/MS