Dehydroepiandrosterone (DHEA); 4-androstene-3,17-dione (AD); and testosterone are androgenic 19-carbon steroids that serve as precursors for other male and female sex hormones. They influence sex-specific and sexually dimorphic processes such as spermatogenesis, muscle growth and neural development. In humans, the adrenal glands primarily produce DHEA, while adrenals and gonads both produce AD. The testes produce 95% of testosterone in males, compared to 50% by the ovaries and adrenal precursors in females. Quantifying steroids in biological matrices is challenging because methods must be specific and sensitive enough to detect relatively low-abundance endogenous analytes. Additionally, steroid concentrations change depending on an individual’s developmental stage. Frey et al. (2016) describe the quantitative method they used to analyze DHEA, AD and testosterone in human serum by stable-isotope dilution liquid chromatography–high-resolution mass spectrometry (LC-HRMS).1
The investigators added an internal standard solution containing 10 pg/μl testosterone, 10 pg/μl AD and 50 pg/μl DHEA in methanol (20 μl) to 100 μl samples of double-charcoal stripped serum or serum. Before injecting the preparation, they added 20 μl of Girard’s Reagent P (GP) to derivatize the keto groups of testosterone, AD and DHEA, to improve ionization efficiency using electrospray ionization. Frey et al. then processed the prepared samples on an UltiMate 3000 quaternary ultra-performance LC system (Thermo Scientific) equipped with a refrigerated autosampler (6°C) and a column heater (60°C) and coupled to a Q Exactive Plus HR mass spectrometer (Thermo Scientific). They performed inter-day and intra-day method validation using LC-HRMS with qualifying product ions and further validated their results by comparing steroid levels from 100 μl of serum from young subjects versus older subjects.
Overall, DHEA and testosterone formed mono-GP products and AD, containing two ketones, derivatized as both the mono-GP and bis-GP derivatives. All androgen GPs analyzed produced an intense product ion still containing the intact steroid ring with a loss derived from the GP derivative after high energy collision dissociation with fragmentation.
To demonstrate the validity of this method, the authors analyzed steroids from 19 subjects: nine men and one woman between 28 years of age and 49 years of age, and eight women and one man between 61 years of age and 81 years of age. Targeted analysis found a predicted drop in testosterone for the age difference and sex distribution of the two sets of participants and a decrease in DHEA.
Frey et al. conclude that derivatization may be a useful way to increase the ion current, which would facilitate multiplexing.
1. Frey, A.J., et al. (2016) “Validation of highly sensitive simultaneous targeted and untargeted analysis of keto-steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry,” Steroids, 116 (pp. 60–66), doi: 10.1016/j.steroids.2016.10.003.