Profiling oestrogens and testosterone in human urine by stable isotope dilution/benchtop gas chromatography–mass spectrometry

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• 作者：Philipp Hoffmann ; Michaela F. Hartmann ; Thomas Remer ; Klaus-Peter Zimmer ; Stefan A. Wudy
• 关键词：Oestrogen ; Testosterone ; Gas chromatography ; Mass spectrometry ; Urine ; Stable isotope
• 刊名：Steroids
• 出版年：2010
• 出版时间：12 December 2010
• 年：2010
• 卷：75
• 期：13-14
• 页码：1067-1074
• 全文大小：598 K

文摘

Oestrogens, such as oestrone (E₁), 17β-oestradiol (E₂), oestriol (E₃) and their biologically active metabolites 2-methoxyoestrone (2-MeOE₁), 2-hydroxyoestradiol (2-OHE₂) 16-ketooestradiol (16-OE₂), 16-epioestriol (16-epiE₃), as well as testosterone (T) play an important role in physiological and pathological developmental processes during human development. We therefore aimed at developing an isotope dilution/bench top gas chromatography–mass spectrometry (ID/GC–MS) method, based on benchtop GC–MS, for the simultaneous determination (‘profiling’) of the above analytes in children. The method consisted of equilibration of urine (5 ml) with a cocktail containing stable isotope-labelled analogues of the analytes as internal standards ([2,4-²H₂]E₁, [2,4,16,16-²H₄]E₂, [2,4,17-²H₃]E₃, [16,16,17-²H₃]T, [1,4,16,16-²H₄]2-MeOE₁, [1,4,16,16,17-²H₅]2-OHE₂, [2,4,15,15,17-²H₅]16-OE₂ and [2,4-²H₂]16-epiE₃). Then, solid-phase extraction (C₁₈ cartridges), enzymatic hydrolysis (sulphatase from Helix pomatia (type H-1)), re-extraction, purification by anion exchange chromatography and derivatisation to trimethylsilyl ethers followed. The samples were analysed by GC–MS (Agilent GC 6890N/5975MSD; fused silica capillary column 25 m × 0.2 mm i.d., film 0.10 μm). Calibration plots were linear and showed excellent reproducibility with coefficients of determination (r²) between 0.999 and 1.000. Intra- and inter-assay coefficients of variation (CV) were <2.21 % for all quantified metabolites. Sensitivity was highest for 2-OHE₂ (0.25 pg per absolute injection: signal-to-noise ratio (S/N) = 3) and lowest for 16-epiE₃ (2 pg per absolute injection: S/N = 2.6), translating into corresponding urine sample analyte concentrations of 0.025 ng ml⁻¹ and 0.2 ng ml⁻¹, respectively. Accuracy – determined in a two-level spike experiment – showed relative errors ranging between 0.15 % for 16-OE₂ and 11.63 % for 2-OHE₂. Chromatography showed clear peak shapes for the components analysed. In summary, we describe a practical, sensitive and specific ID/GC–MS assay capable of profiling the above-mentioned steroids in human urine from childhood onwards.