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.