Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays

Abstract

Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined with the multi-steroid LC-MS(2) method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all good. Instrument sensitivity was in the range of 55-530 pg/mL (LLOQ).

Salivary testosterone may not serve as a screening test in the diagnosis of biochemical hyperandrogenism

“…Another immunoenzymatic test, ADVIA Centaur XP, proved to be more effective when testing saliva specimens for the measurement of testosterone level . The cleanup method combined with the LC–MS/MS increases the quality of quantification of steroid hormones and may create an opportunity to increase the quality of the Salimetrics method …”

Effects of antihistamines on the H295R steroidogenesis – Autocrine up-regulation following 3β-HSD inhibition

“…Steroid extraction, cleanup and LC-MS/MS analysis Steroids were extracted and analysed according to Weisser et al (2016). To each eppendorf tube containing 950 µL cell medium 50 µL of 0.1 µg/mL internal standard solution containing deuterated steroid analogues was added.…”

“…The glass tubes were then vortexed and frozen for another 10 min and centrifuged at ~1500 G for 10 min. Supernatant was transferred to 1.5 mL LC-vials and evaporated under a gentle stream of nitrogen at 60 °C until ~0.5 mL left (Weisser et al, 2016). Finally, milli-Q water was added up to 1.0 mL.…”

“…Online clean-up Liquid Chromatography (LC) followed by mass spectrometry (MS) was used to quantify steroid hormones (Weisser et al, 2016). A binary 1100 Agilent HPLC pump and a binary 1290 Agilent Infinity Series system were used in combination for online clean-up and chromatographic separation of all steroid hormones.…”

“…In front of the analytical column a guard column (C 18 , 2.1 mm, Phenomenex, USA) was placed, and they were connected to the TTC switching valve and a MS switching valve. To the TCC switching valve a 1290 pump was connected (Weisser et al, 2016). A mass spectrometer (AB SCIEX 4500 QTRAP) was used for detection, provided with an atmospheric pressure chemical ionization Turbo V source.…”