Simultaneous Quantification of 22 Phenolic Compounds in Human Serum Using 96-Well HybridSPE-PPT and Dansyl Derivatization-Based LC-MS/MS

Environmental phenolic endocrine-disrupting chemicals (EDCs) are ubiquitous in the environment and human matrices, raising growing concerns regarding maternal and child health. Here, we present a sensitive analytical method for the simultaneous quantification of 22 phenolic compounds—including 7 bisphenols, 2 alkylphenols, 11 pharmaceutical and personal care product-related phenols, and 2 endogenous estrogens—in human serum. The method combines enzymatic deconjugation, 96-well plate-based protein precipitation and phospholipid removal using HybridSPE technology with dansyl chloride derivatization and liquid chromatography–tandem mass spectrometry, requiring only 100 μL of serum per sample. Validation using fetal bovine serum as a surrogate matrix yielded limits of detection of 0.023–0.227 ng/mL, recoveries of 86–114%, relative standard deviations below 20%, and matrix effects ranging from −35% to −2%. The method was applied to 30 serum samples from pregnant women in southern China, with nine analytes—including bisphenol A, triclosan, and endogenous estrogens—detected in at least 70% of samples. The median concentrations of the target compounds following enzymatic deconjugation ranged from below limits of detection to 28.4 ng/mL (for estrone). This method enables the simultaneous analysis of environmental phenols and endogenous estrogens in limited-volume human serum, supporting biomonitoring studies.