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| Determination of eleven steroid hormones in human urine by ultra-high performance liquid chromatography-tandem mass spectrometry |
| WANG Heng1, MA Xiaolong2
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1. Zhoushan Center for Disease Control and Prevention (Zhoushan Institute of Public Health Supervision), Zhoushan, Zhejiang 316021, China;
2. School of Public Health, China Medical University, Shenyang, Liaoning 110122, China |
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Abstract Objective To establish a method for the determination of 11 steroid hormones in human urine by ultra-high performance liquid chromatography-tandem mass spectrometry through optimizing sample pretreatment, chromatographic and mass spectrometric conditions. Methods Urine samples were enzymatically hydrolyzed overnight with β-glucuronidase, and then extracted by vortex oscillation using methyl tert-butyl ether. The organic phase was collected after centrifugation, dried with nitrogen gas, redissolved in aqueous acetonitrile solution, and centrifuged for subsequent testing. The extraction efficiency and response intensity were improved by optimizing the parameters of sample pretreatment, chromatography and mass spectrometry. Standard curves of 11 steroid hormones were plotted, and the limits of detection, limits of quantification, average spiked recovery rate and relative standard deviation (RSD) were calculated. Results The optimal sample pretreatment procedure involved a single extraction step with 3 mL of methyl tert-butyl ether. Under the positive ion mode, 0.1% acetic acid aqueous solution/acetonitrile was selected as the chromatographic mobile phase system. The quantitative ion pairs of 271.2>133.1 and 255.2>133.1 were selected for the detection of estrone and 17β-estradiol, respectively. After methodological optimization, the matrix effects of the 11 steroid hormones ranged from 93.9% to 108.2%, and the internal standard method was applied for quantitative analysis. Pregnenolone exhibited a good linear relationship in the range of 0.5-100 ng/mL, with the limits of detection and limits of quantification of 0.030 ng/mL and 0.100 ng/mL, respectively. The other 10 steroid hormones had good linear relationships in the range of 0.05-100 ng/mL, with the limits of detection and limits of quantification of 0.003 ng/mL and 0.010 ng/mL, respectively. The average spiked recovery rates of the 11 steroid hormones ranged from 80.9% to 169.9%, with RSD ranging from 0.3% to 18.6%. All 11 steroid hormones were detected in 32 urine samples collected from healthy volunteers. Conclusions The optimized method exhibits a slight matrix effect, with the limit of detection, limit of quantification, accuracy and precision all meeting the detection requirements. It can be applied to the determination of 11 steroid hormones in human urine.
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Received: 30 March 2026
Revised: 01 June 2026
Published: 21 August 2026
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[1] Dlugash G,Rauh M,Carré J M,et al.Multicenter comparison of LC-MS/MS,radioimmunoassay,and ELISA for assessment of salivary cortisol and testosterone[J/OL].Psychoneuroendocrinology,2026,183[2026-06-01].http://doi.org/10.1016/j.psyneuen.2025.107618.
[2] 陈冬婵,金薇.性早熟女童糖脂代谢与性激素水平分析[J].预防医学,2022,34(9):951-954.
[3] Zhan X,Han L S,Qiu W J,et al.Steroid profile in dried blood spots by liquid chromatography tandem mass spectrometry:application to newborn screening for congenital adrenal hyperplasia in China[J/OL].Steroids,2022,185[2026-06-01].http://doi.org/10.1016/j.steroids.2022.109056.
[4] 黄宪章. 液相色谱-串联质谱临床推广与应用面临挑战[J].中华检验医学杂志,2021,44(8):661-663.
[5] 张劢,李艳,刘炳锐,等.液质联用技术同时测定血清中12种类固醇激素[J].中国医药科学,2025,15(9):150-153.
[6] Chotboon C,Salang L,Buppasiri P,et al.Association between urine and serum estradiol levels in in vitro fertilization cycles[J/OL].Sci Rep,2022,12(1)[2026-06-01].http://doi.org/10.1038/s41598-022-08292-z.
[7] Zhou Y Q,Cai Z W.Determination of hormones in human urine by ultra-high-performance liquid chromatography/triple-quadrupole mass spectrometry[J/OL].Rapid Commun Mass Spectrom,2020,34(S1)[2026-06-01].http://doi.org/10.1002/rcm.8583.
[8] Rakete S,Schubert T,Vogeser M.Semi-automated serum steroid profiling with tandem mass spectrometry[J].J Mass Spectrom Adv Clin Lab,2023,(27):40-48.
[9] Desai R,Harwood D T,Handelsman D J.Simultaneous measurement of 18 steroids in human and mouse serum by liquid chromatography-mass spectrometry without derivatization to profile the classical and alternate pathways of androgen synthesis and metabolism[J].Clin Mass Spectrom,2019,(11):42-51.
[10] 朱传新,郑艳容.超高效液相色谱-串联三重四极杆质谱法同时分析尿中4种内源性雄激素和孕激素[J].中国卫生检验杂志,2014,24(7):927-929.
[11] 夏冰洋,马亚萍,李楠楠,等.超高效液相色谱-串联质谱法同时测定人尿中5种甾体激素[J].扬州大学学报(农业与生命科学版),2016,37(3):30-36.
[12] Jia Y J,Liu X L,Xu L,et al.Liquid chromatography-tandem mass spectrometry measurement of 26 steroid hormones in human serum and plasma samples[J].J Sep Sci,2021,44(12):2358-2370.
[13] 冯媛媛,田冶,尹利辉,等.液质联用分析技术中的基质效应[J].中国新药杂志,2024,33(10):1009-1020.
[14] Food and Drug Administration.Bioanalytical method validation guidance for industry[EB/OL].[2026-06-01].https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry.
[15] Marcos J,Renau N,Casals G,et al.Investigation of endogenous corticosteroids profiles in human urine based on liquid chromatography tandem mass spectrometry[J].Anal Chim Acta,2014,812:92-104.
[16] Bain J T,Shaheen F,Prete A,et al.Comprehensive quantitative urinary steroid profiling of 29 steroids using liquid chromatography-tandem mass spectrometry[J/OL].Anal Sci Adv,2026,7(1)[2026-06-01].http://doi.org/10.1002/ansa.70087. |
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