Analytical Sciences

Simultaneous Determination of Seven Neuroactive Steroids Associated with Depression in Rat Plasma and Brain by High Performance Liquid Chromatography–Tandem Mass Spectrometry

Youqiong WANG,*1,*2 Lipeng TANG,*1,*3 Wei YIN,*4 Jiesi CHEN,*1 Tiandong LENG,*1 Xiaoke ZHENG,*1 Wenbo ZHU,*1 Haipeng ZHANG,*1 Pengxin QIU,*1 Xiaoxiao YANG,*1 Guangmei YAN,*1 and Haiyan HU*5

*1 Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-Sen University, No. 40, Zhongshan 2nd Road, Guangzhou, GD 510080, China
*2 Department of Pharmacy, The First Affiliated Hospital of Guangxi University of Chinese Medicine, No. 89-9 Dongge Road, Nanning, GX 530023, China
*3 Department of Pharmacology of Traditional Chinese Medicine, Guangdong Province’s Traditional Chinese Medical Hospital, No. 111 Dade Road, Guangzhou, GD 510006, China
*4 Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-Sen University, No. 40, Zhongshan 2nd Road, Guangzhou, GD 510080, China
*5 School of Pharmaceutical Sciences, Sun Yat-Sen University, No. 132 Outer Ring East Road, Higher Education Mega Center, Guangzhou, GD 510006, China

Sensitive and specific biomarkers are required for the diagnosis and treatment of depression because the existing diagnostic criteria are subjective and could produce false positives or negatives. Some endogenous neuroactive steroids that have shown either antidepressant effects or concentration changes in individuals with depression could provide potential biomarkers. In this study, a simple and specific method was developed to simultaneously determine seven endogenous neuroactive steroids in biological samples: cortisone, cortisol, dehydroepiandrosterone, estradiol, progesterone, pregnenolone, and testosterone. After liquid–liquid extraction, chromatographic separation was achieved on a C18 column with gradient elution using water–methanol at a flow rate of 300 μL min⁻¹. Detection and quantitation were performed by tandem mass spectrometry with atmospheric pressure chemical ionization and selected reaction monitoring. Plasma and brain neuroactive steroid levels were then determined in control rats and rats exposed to forced swimming, a classical rodent model of depression. The results showed that the plasma concentrations of testosterone, pregnenolone, and progesterone significantly increased in rats exposed to the forced swimming test. In contrast, brain homogenate levels of cortisol, estradiol, and progesterone decreased, while pregnenolone levels were elevated in this model of depression. In conclusion, a new method to quantify neuroactive steroids was successfully developed and applied to their investigation in rat plasma and brain. The findings of this study indicated that plasma testosterone, pregnenolone, and progesterone levels could provide potential biomarkers for the diagnosis and treatment of depression.

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