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ABSTRACT Rationale Phenolic endocrine-disrupting chemicals (EDCs), including alkylphenols (APs) and bisphenols (BPs), pose significant environmental and public health concerns due to their widespread presence in aquatic ecosystems and potential toxicity at trace concentrations. This study developed and validated a sensitive analytical method for the simultaneous determination of 16 phenolic EDCs in environmental waters. Methods The method involves sample acidification to pH 2, hydrophilic–lipophilic balance (HLB), solid-phase extraction (SPE), and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) analysis with negative electrospray ionization (ESI−). Systematic optimization revealed that an alkaline mobile phase (methanol/0.1% ammonia) significantly enhanced the ionization efficiency of phenolic compounds, improving sensitivity by approximately an order of magnitude compared to acidic conditions. Chromatographic separation was optimized using an ACQUITY UPLC BEH C18 column, which provided superior peak shapes and improved retention of hydrophobic compounds. Results Acidification of samples prior to SPE was critical for maximizing analyte recovery (57.9%–94.2%). The validated method demonstrated outstanding analytical performance, achieving low detection limits (0.01–0.4 ng/L), excellent linearity (R = 0.991–0.999) across a broad concentration range (1.0–50.0 μg/L) and high precision (RSDs = 1.3%–13.6%). Application of the method to environmental samples collected from rivers, wastewater treatment plants, and landfill leachates identified eight target compounds, with alkylphenols detected at higher concentrations than BPs. Conclusion This reliable and robust method provides a valuable tool for monitoring trace levels of phenolic EDCs in diverse aquatic environments, supporting technically comprehensive environmental assessments and regulatory compliance.

ABSTRACT Background Tris(2-carboxyethyl)phosphine (TCEP) is a commonly used laboratory reagent for the purposes of reducing disulfide bonds. TCEP is often preferred over alternative reducing reagents, such as dithiothreitol (DTT), due to its strong redox potential, effectiveness in a broad pH range, and handling considerations. Despite this, many side reactions involving TCEP have been poorly categorized and understood. Finding Utilizing a combination of gel electrophoresis and mass spectrometry techniques, we have discovered a unique and novel mechanism by which TCEP engages in the hydrolysis of proteins. This behavior has been observed to be site specific to the carboxyl terminus of aspartic acid residues. Conclusion Cleavage at this location is completely independent of the reduction of disulfide bonds in proteins due to the observance of this phenomenon in proteins lacking disulfide bonds, and some lacking cysteines altogether. While the specific chemistry of this hydrolysis is unknown, this discovery has potentially wide-ranging impacts as a sample preparation tool for mass spectrometry analysis, as well as being cautionary towards other common laboratory uses of this ubiquitous reagent where it could potentially cause unwanted hydrolysis of proteins being studied. Impact The broader analytical implications of this finding are that many previously unknown origin artifacts can now be properly attributed to a source, particularly in areas such as proteomic sample preparation, redox chemistries studies, and similar. This also expands our knowledge of TCEP chemistry beyond simply a disulfide bond reductor, offering both methodological opportunities for peptide mapping and important cautionary implications for biochemical sample treatment.