Journal article

Publication year: 2010

Pages: 1067-1074

Journal: Steroids

Volume number: 75

Issue number: 13-14

ISSN: 0039-128X

DOI Link: https://doi.org/10.1016/j.steroids.2010.06.014

Publisher: Elsevier

Abstract: 
Oestrogens, such as oestrone (E(1)), 17 beta-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-(2)H(2)]E(1), [2,4,16,16-(2)H(4)]E(2), [2,4,17-(2)H(3)]E(3), [16,16,17-(2)H(3)]T, [1,4,16,16-(2)H(4)]2-MeOE(1), [1,4,16,16,17-(2)H(5)]2-OHE(2), [2,4,15,15,17-(2)H(5)]16-OE(2) and [2,4-(2)H(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 25m x 0.2 mm i.d., film 0.10 mu m). 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. (C) 2010 Elsevier Inc. All rights reserved.

Harvard Citation style: Hoffmann, P., Hartmann, M., Remer, T., Zimmer, K. and Wudy, S. (2010) Profiling oestrogens and testosterone in human urine by stable isotope dilution/benchtop gas chromatography-mass spectrometry, Steroids, 75(13-14), pp. 1067-1074. https://doi.org/10.1016/j.steroids.2010.06.014

APA Citation style: Hoffmann, P., Hartmann, M., Remer, T., Zimmer, K., & Wudy, S. (2010). Profiling oestrogens and testosterone in human urine by stable isotope dilution/benchtop gas chromatography-mass spectrometry. Steroids. 75(13-14), 1067-1074. https://doi.org/10.1016/j.steroids.2010.06.014