Determination of atrazine and propazine metabolites deemed endocrine disruptors in human seminal plasma by LC-ESI-MS/MS

Background: The increasing prevalence of male infertility and the declining trend in sperm quality has been associated to compounds known as "endocrine-disruptors". The proven endocrine-disrupting effects of atrazine and propazine herbicides led us to conduct long-term research based on highly accurate specific analytical methods with a view to confirming the suspected association. Among the proposed developments was a sensitive analytical method for the simultaneous determination of three metabolites of atrazine and propazine. Results: In this work, the method was for first time used for the chromatographic separation and determination of deethyl- and deisopropyl-atrazine (DEA and DIA, respectively) and propazine-2-hydroxy (PP-2OH) in human seminal plasma by LC-ESI-MS/MS using deuterated atrazine (d5-AT) as internal standard (IS). Chromatographic and mass spectrometric conditions such as the mobile phase composition and flow-rate, injected volume, dry gas source temperature and flow-rate, nebulizer pressure and capillary voltage were all carefully optimized. Analytes were identified and quantified by using the multiple reaction monitoring (MRM) mode as applied to positive ions ([M + H](+)). Transitions at three different m/z values for each analyte were selected from precursor ions, and the 212.1 -> [128](+), 188.1 -> [146](+) and 174.1 -> [68.1](+) transitions for PP-2OH, DEA and DIA, respectively, were found to be quantitative. The proposed method was validated in terms of precision (repeatability and reproducibility), linear range (10-240 ng mL(-1)), limit of detection (150-210 pg mL(-1)), and quantification (500-700 pg mL(-1)), recovery, accuracy and matrix effects on extracts from variably treated seminal plasma samples. The overall analytical method was successfully applied to human seminal plasma samples from volunteers. PP-2OH was found at concentrations from 1.10 to 11.3 ng mL(-1) in four of the six samples, and so was DIA at 9.60 ng mL(-1) in one. Conclusions: These results are suggestive of bioaccumulation of the target analytes in humans. Untargeted analytes including suspected parent molecules (atrazine and propazine) and other ions [viz., deethyldeisopropyl-atrazine (DD) and diamino-s-chlorotriazine (DACT)] were also detected under the working conditions used. These results may open up new prospects for as yet very incipient research into the bioaccumulation of endocrine disruptors in seminal plasma.