Determination of eleven steroid hormones in human urine by ultra-high performance liquid chromatography-tandem mass spectrometry
WANG Heng1, MA Xiaolong2
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
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.
王恒, 马小龙. 超高效液相色谱-串联质谱法检测人体尿液11种甾体类激素[J]. 预防医学, 2026, 38(8): 854-858,864.
WANG Heng, MA Xiaolong. Determination of eleven steroid hormones in human urine by ultra-high performance liquid chromatography-tandem mass spectrometry. Preventive Medicine, 2026, 38(8): 854-858,864.
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