Endocrine Abstracts

Background: The classical steroidogenic route to the synthesis of dihydrotestosterone in Leydig cells involves delta-5 and delta-4 precursors. Besides, the “backdoor pathway”, encompassing progesterone metabolites, is gaining increasing interest in fetal development and men pathophysiology. Nowadays, liquid chromatography/tandem mass spectrometry (LC-MS/MS) is the ideal technique to simultaneously quantify large panels of steroids with elevated sensitivity and specificity.

Aim: We developed and validated a LC-MS/MS method measuring 20 among androgens, precursors and metabolites pertaining to both the classical and the backdoor pathways in the steroidogenic mouse Leydig tumor cell line 1 (mLTC1).

Methods: Steroidogenesis of serum-starved mLTC1 cells was induced by 100µM choriogonadotropin (hCG). Steroids released in 500µL cell medium were extracted with 2mL N-hexane:ethyl-acetate (8:2) spiked with 100µL internal standards in methanol. The organic phase was dried and samples were reconstituted with 100µL of 75% methanol before 10µL were analyzed. The platform included the Serie200 HPLC (Perkin Elmer) and the API-4000 QTrap (Sciex) operated in multiple reaction monitoring mode. 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone (21OH-progesterone), testosterone, androstenediol, 17OH-progesterone, 17OH-pregnenolone, dehydroepiandrosterone, androstanedione, epitestosterone, dihydrotestosterone, 17OH-dihydroprogesterone, progesterone, androstanediol, androsterone, pregnenolone, 5α-dihydroprogesterone, 17OH-allopregnanolone and allopregnanolone were separated in 13-min gradient operated on a Luna C8 100x3 mm, 3 µm column, equipped with 4x2.0 mm guard column (Phenomenex), termostated at 45°C, using 100µM ammonium fluoride and methanol as mobile phases. Analytes were revealed by quantitative and qualitative transitions and quantified by isotopic dilution.

Results: Isobaric compounds were separated to ensure selectivity. The dynamic range was between 3 and 5 orders of magnitude. Functional sensitivity was between 0.012 and 38 nmol/l. Intra-assay and inter-assay imprecision and trueness, valued at low, medium and high levels, were below 9.1 and 10.0%, and within 93.4-122.0%, respectively. Recovery and matrix factor were within 55.6-104.1% and 76.4-106.3%, respectively. Levels (nmol/l) of 16OH-progesterone (20.7±4.2), 11-deoxycortisol (0.924±0.115), androstenedione (727±89), 11-deoxycorticosterone (3.69±0.40), testosterone (52.8±10.1), 17OH-progesterone (6.58±0.75), androstanedione (774±164), epitestosterone (26.1±4.4), dihydrotestosterone (9.61±2.51), progesterone (4.79±0.60), androsterone (379±80) and 17OH-allopregnanolone (4.30±0.58) were within the measurement range. Levels of 17OH-dihydroprogesterone (0.844±0.107), androstanediol (21.5±5.9) and dihydroprogesterone (0.791±0.136) were slightly above the limit of detection, whereas other compounds were undetectable.

Conclusions: We validated a LC-MS/MS method including a broad number of androgens, precursors and metabolites. Medium secretion levels from mLTC1 cells were measured effectively for most of the steroids, however, levels of some neutral and delta-5 steroids were below method’s sensitivity. Nonetheless, we provided a powerful tool to simultaneously characterize the gonadal classical and backdoor steroidogenic pathways.