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

Abstract

Oestrogens, such as oestrone (E 1), 17β-oestradiol (E 2), oestriol (E 3) and their biologically active metabolites 2-methoxyoestrone (2-MeOE 1), 2-hydroxyoestradiol (2-OHE 2) 16-ketooestradiol (16-OE 2), 16-epioestriol (16-epiE 3), 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- 2H 2]E 1, [2,4,16,16- 2H 4]E 2, [2,4,17- 2H 3]E 3, [16,16,17- 2H 3]T, [1,4,16,16- 2H 4]2-MeOE 1, [1,4,16,16,17- 2H 5]2-OHE 2, [2,4,15,15,17- 2H 5]16-OE 2 and [2,4- 2H 2]16-epiE 3). Then, solid-phase extraction (C 18 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 ( r 2) 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-OHE 2 (0.25 pg per absolute injection: signal-to-noise ratio (S/N) = 3) and lowest for 16-epiE 3 (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-OE 2 and 11.63% for 2-OHE 2. 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.