Measurement of sex steroids in murine blood and reproductive tissues by liquid chromatography-tandem mass spectrometry

Accurate measurement of sex steroids is essential to evaluate mouse models for human reproductive development and disorders. The recent advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays that match the sensitivity of steroid immunoassay could overcome problems arising from the limited specificity of steroid immunoassay. In this current study we validate a LC-MS/MS assay for the measurement of key sex steroids from murine serum and reproductive tissues. The assay gave excellent dilutional linearity (r²≥0.98) and reproducibility (CV≤10% of replicate samples) in serum and reproductive tissues with sensitive quantitation limits; testosterone (T; 2pg), dihydrotestosterone (DHT; 10pg), 5α-androstane-3α,17β-diol (3αDiol; 40pg), 5α-androstane-3β,17β-diol (3βDiol; 40pg), estradiol (E2; 0.5pg) and estrone (E1; 0.3pg). Using 0.1mL sample, T was the only consistently detectable steroid (detection limit 20pg/ml) in both male and female mouse serum. In the testis, T and DHT were quantifiable as were both diols at relatively high levels. Prostatic T levels were low and DHT was determined to be the most abundant androgen in this tissue. Uterine and ovarian levels of E2, E1 and T were measurable, with levels varying according to estrous cycle stage. Hence, we demonstrate that this LC-MS/MS method has the sensitivity, specificity and multi-analyte capability to offer accurate steroid profiling in mouse serum and reproductive tissues.