| Summary: | The 8-iso-prostaglandin F<sub>2α</sub> (8-iso-PGF<sub>2α</sub>) biomarker is used as the gold standard for tracing lipid oxidative stress in vivo. The analysis of urinary 8-iso-PGF<sub>2α</sub> is challenging when dealing with trace amounts of 8-iso-PGF<sub>2α</sub> and the complexity of urine matrixes. A packed-fiber solid-phase extraction (PFSPE)–coupled with HPLC-MS/MS–method, based on polystyrene (PS)-electrospun nanofibers, was developed for the specific determination of 8-iso-PGF<sub>2α</sub> in urine and compared with other newly developed LC-MS/MS methods. The method, which simultaneously processed 12 samples within 5 min on a self-made semi-automatic array solid-phase extraction processor, was the first to introduce PS-electrospun nanofibers as an adsorbent for the extraction of 8-iso-PGF<sub>2α</sub> and was successfully applied to real urine samples. After optimizing the PFSPE conditions, good linearity in the range of 0.05–5 ng/mL with R<sup>2</sup> > 0.9996 and a satisfactory limit of detection of 0.015 ng/mL were obtained, with good intraday and interday precision (RSD < 10%) and recoveries of 95.3–103.8%. This feasible method is expected to be used for the batch quantitative analysis of urinary 8-iso-PGF<sub>2α</sub>.
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