6533b7d6fe1ef96bd1265bbe

RESEARCH PRODUCT

Gas chromatography/mass spectrometry of catechol estrogens

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Abstract Catecholestrogens (CCEs), namely 2- or 4-hydroxyestradiol and hydroxyestrone, are highly polar, reactive, and extremely labile estrogen metabolites in many experimental conditions. For these reasons, indirect assay methods mainly have been used. Some experimental evidence suggests that CCEs are synthesized and biologically active mostly in target cells. At this level, unfortunately, the indirect assays cannot be used. We present a method of gas Chromatographic/mass spectral (GC/MS) analysis for the identification of individual CCEs; the major fragmentation ions of authentic estrogen standards as trimethylsilylether derivatives, and the MS patterns of the major CCEs, namely, 2-hydroxyestradiol and hydroxyestrone, are included. Few examples of CCEs detected in human breast cancer tissues and in breast cyst fluids are reported. Sample extracts were submitted to reversed-phase, high-performance liquid chromatography (RP-HPLC) and were quantified by “on line” electrochemical (EC) detection; thereafter, either crude extracts or single eluted peaks were submitted to GC/MS, by which detection limits of less than 5 pmol were attained. As expected, the molecular ion was the most relevant molecule in all but one case. On the contrary, the other relative intensities of major fragmentation ions M −15, M −30, M −90, and M −15 + (−90) were unevenly distributed, although represented in the majority of cases. In all cases, the GC/MS of peak fractions, purified by RP-HPLC and UV detection, confirmed the results of liquid Chromatographic analysis combined with EC detection. In contrast, GC/MS of crude extracts was not equally satisfactory. Comparison of a liquid chromatography system with EC detection and the GC/MS approach revealed some inconsistency in quantitation of individual CCEs. Despite the fact that RP-HPLC with EC detection is sensible and versatile enough to analyze a number of free estrogen metabolites, the results of present study strongly suggest the need for a preliminary sample purification for CCE identification by GC/MS. This represents an essential requirement for intratissue concentration studies.