(T1130-01-06) Species and Gender-Related Variations in Dehydroepiandrosterone Sulfate Biliary Excretion: A Comparative LC-MS/MS Analysis

Purpose: Dehydroepiandrosterone sulfate (DHEAS), the sulfate metabolite of dehydroepiandrosterone (DHEA), is an important endogenous compound (a precursor to sex hormones). Epidemiology studies showed the correlation between DHEAS level fluctuations and various health conditions (1-4). Biliary excretion, as an integral component of enterohepatic recirculation (EHR), represents a crucial pathway for the elimination of endogenous compounds and xenobiotics. This pathway has been proposed to influence DHEAS profile modification and facilitate excretion, thereby affecting systemic hormone levels (5, 6). Although species and gender differences in plasma DHEAS concentrations are well-documented (7-9). DHEAS levels in bile samples across male and female subjects remain poorly characterized. This study aimed to develop a validate LCMS method of DHEAS quantification in bile and blood and investigate interspecies and gender-related variations in DHEAS biliary excretion.

Methods: Charcoal-stripped whole blood and bile samples from rats were utilized to mimic steroid-free matrices for the method validation (10). An analytical method was developed using the simple protein precipitation method with deuterated internal standard (DHEAS-D5) using 5 µL aliquots of all sample matrices. All analysis was performed under API 7500 Qtrap mass spectrometer equipped with chromatographic system (UPLC) using an HSS-T3 column for separation and quantification. Bile and whole blood samples were collected from male and female rats, as well as bovine and porcine bile samples, to quantify DHEAS and identify gender-specific patterns across species.

Results: The assay was successfully validated according to the US Food and Drug (FDA) guideline (11) for quantifying the DHEAS in all biological matrices. With simple precipitation method, it mostly demonstrated the good recovery from all matrices (80.0 – 110.0%) with minimal matrix effect (90.0 – 110%). This assay achieved the linearity range in nanomolar and mostly showed the precision (%CV) and accuracy (%bias) of intra- and inter-day within ± 20%. The lower limit of quantification (LLOQ) was established at 0.24 nmole/L. Significant species differences in biliary DHEAS excretion were observed, except between porcine and bovine samples. Furthermore, the findings revealed substantial DHEAS concentrations in bile samples, with consistently higher levels observed in females across all examined species. These results suggest that both species and gender are influential factors in DHEAS biliary excretion patterns. In contrast, whole blood DHEAS levels demonstrated no significant differences between males and females’ rats, with most concentrations falling below the LLOQ.

Conclusion: The developed LC-MS/MS method is simple, sensitive, and robustly validated for DHEAS quantification in biological samples. This study is the first to report gender and species differences in DHEAS biliary excretion, providing foundational insights that may enhance understanding of DHEAS profiles in preclinical and clinical research settings.

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Acknowledgements: Rashim Singh is supported by the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health under award number K12TR004522.

Figure 1 (A) Matrix effect and Recovery of DHEAS in blood and bile samples, and (B) Heat plots of precision (%CV) and accuracy (%Bias) of intra-day and inter-day for DHEAS quantification


Figure 2 Quantification of DHEAS in (A) blood and (B) bile comparing between male and female rats (n = 3 per sex). Data are presented as the means ± SD. P-values were determined by unpaired Student's t-test.

Figure 3 Concentration of DHEAS in bile samples obtained from different species comparing between male and female (A) Rats, (B) Bovine, and (C) Porcine (n = 3 per sex). Data are presented as the means ± SD. P-values were determined by unpaired Student's t-test.