Andrology, ANZAC Research Institute, University of Sydney, Sydney, NSW 2139, Australia.
J Steroid Biochem Mol Biol. 2010 Aug;121(3-5):611-8. doi: 10.1016/j.jsbmb.2010.02.001. Epub 2010 Feb 6.
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 (r(2)> or =0.98) and reproducibility (CV< or =10% of replicate samples) in serum and reproductive tissues with sensitive quantitation limits; testosterone (T; 2pg), dihydrotestosterone (DHT; 10pg), 5alpha-androstane-3alpha,17beta-diol (3alphaDiol; 40pg), 5alpha-androstane-3beta,17beta-diol (3betaDiol; 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.
准确测量性激素对于评估人类生殖发育和疾病的小鼠模型至关重要。最近出现的液相色谱-串联质谱（LC-MS/MS）分析方法与类固醇免疫分析的灵敏度相匹配，可以克服类固醇免疫分析特异性有限所带来的问题。在本研究中，我们验证了一种用于测量小鼠血清和生殖组织中关键性激素的 LC-MS/MS 分析方法。该方法在血清和生殖组织中具有出色的稀释线性（r(2)>或=0.98）和重现性（重复样品的 CV<或=10%），具有敏感的定量限；睾酮（T）；二氢睾酮（DHT）；5α-雄烷-3α,17β-二醇（3α二醇）；5α-雄烷-3β,17β-二醇（3β二醇）；雌二醇（E2）和雌酮（E1）。使用 0.1mL 样本，T 是男女小鼠血清中唯一可检测到的类固醇（检测限 20pg/ml）。在睾丸中，T 和 DHT 以及两种二醇均可定量检测，水平较高。前列腺 T 水平较低，DHT 被确定为该组织中最丰富的雄激素。子宫和卵巢中 E2、E1 和 T 的水平可测量，其水平随发情周期阶段而变化。因此，我们证明了这种 LC-MS/MS 方法具有灵敏度、特异性和多分析物能力，可以在小鼠血清和生殖组织中进行准确的类固醇分析。
