Simultaneous determination of phytoestrogens and key metabolites in breast cancer patients’ urine by liquid chromatography–tandem mass spectrometry

A novel, selective and sensitive liquid chromatography–tandem mass spectrometry method has been developed and validated for the simultaneous determination of phytoestrogens and their key metabolites in human urine in this study. This method includes internal standard (IS) screening, analytical sample preparation procedure establishment, and linear range investigation. The analytical sample was extracted by liquid–liquid extraction from urine sample. The phytoestrogens and related key metabolites were separated with Agilent Zorbax Eclipse XDB-C18 chromatographic column using methanol and water as mobile phase. The Quattro premier MICROMASS mass spectrometer in negative ion selected reaction monitoring (SRM) mode using electrospray ionization was applied to detect the phytoestrogens and key metabolites. To validate the developed liquid chromatography–tandem mass spectrometry method, the intra- and inter-day precisions, specificity, sensitivity, reproducibility, and sample detective concentration range were evaluated. This is the first reported phytoestrogens analysis and validation study that demonstrates the feasibility of using liquid chromatography–electrospray ionization mass spectrometry to simultaneously analyze ten analytes including both phytoestrogens and their key metabolites in urine samples collected for epidemiological studies in human.