Dispersive μSPE in a 96-well filtration plate for high-throughput LC–MS/MS quantification of bile acid sulfates in human urine

We report a chemometrically optimized high-throughput analytical workflow integrating filtration-assisted dispersive micro-solid phase extraction (FA-D-μSPE) with LC–MS/MS for quantitative profiling of ten major human bile acid-3-sulfates (BA-S) in urine. BA-S are important urinary metabolites of bile acid detoxification and promising non-invasive biomarkers of hepatobiliary disorders. The proposed workflow implements dispersive μSPE in a 96-well filtration format, enabling in-plate sorbent extraction without centrifugation, magnetic separation, or manual phase separation. Method development was guided by sequential experimental designs (Plackett–Burman, central composite, and Box–Behnken), allowing statistically guided optimization of chromatographic separation, mass spectrometer settings, and extraction conditions. A custom 3D-printed dispenser enabled fast and reproducible in-house loading of extraction sorbent into filtration plates and facilitated flexible customization of sorbent chemistry. Compared with conventional cartridge-based or well plate SPE, FA-D-μSPE reduces solvent consumption, processing time, and consumable costs while maintaining robust analytical performance. Due to the endogenous presence of BA-S in human urine, method validation was performed using rat urine as a surrogate matrix. The method was validated according to ICH M10 guidelines, demonstrating linearity over 10–4000 nmol L -1 , high accuracy and precision, and controlled matrix effects. The proposed FA-D-μSPE LC–MS/MS method provides a robust and cost-efficient strategy for rapid quantitative bioanalysis of urinary BA-S and may serve as a framework for clinical and epidemiological studies.