Polyacrylonitrile/polypyrrole nanofiber-filled packed-fiber solid-phase extraction coupled with HPLC-FLD for matrix-interference-resistant determination of urinary pteridines and its application in autism spectrum disorder research

PAN/PPy NFs are first applied to PFSPE-HPLC-FLD for urinary pteridines detection. • PAN/PPy NFs adsorb pteridines via synergistic electrostatic, H-bond and π-π forces. • The optimized method gives negligible matrix effects (−6.6%–8.2%) for urine samples. • The sensitive, robust method was fully validated for clinical urine sample analysis. Pteridines are associated with neuroinflammation and oxidative stress, the core pathological processes of autism spectrum disorder (ASD). Urinary neopterin (NPT), biopterin (BPT), and isoxanthopterin (IXP) are promising non-invasive biomarkers for children with ASD. However, matrix interference from urinary salts and metabolites severely impedes trace pteridine detection, while mainstream analytical methods either require cumbersome derivatization or sacrifice sensitivity for dilution to mitigate matrix effects (ME). This study reports the first application of polyacrylonitrile/polypyrrole nanofibers (PAN/PPy NFs) for the determination of pteridines in urine by packed-fiber solid-phase extraction (PFSPE) coupled with HPLC-FLD, without derivatization. XPS, FTIR and fluorescence experiments confirmed pteridine adsorption on PAN/PPy NFs via synergistic electrostatic attraction, hydrogen bonding and π-π stacking. Validation showed: ME -6.6%–8.2%, R 2  > 0.995, LOD s 0.8–5.3 ng/mL, LOQ s 2.8–17.6 ng/mL, and RSD s < 9.7%. AGREE (0.61) and BAGI (72.5) verified its compliance with green analytical chemistry principles. Analysis of 33 ASD and 34 healthy urine samples revealed significantly elevated NPT levels in the ASD group ( p  = 0.011). This efficient, sensitive and green PFSPE-HPLC-FLD method eliminates urinary matrix interference without derivatization, overcoming key limitations of conventional approaches. It provides a reliable tool for urinary pteridine assays in ASD research and supports ASD pathogenesis investigation and exploratory clinical studies. The figure shows PAN/PPy-PFSPE for matrix effect elimination from ASD children's urine. PAN/PPy NFs sorbent, with high porosity and multiple interaction sites, mediates pteridine adsorption via synergistic electrostatic attraction, hydrogen bonding and π-π stacking, enabling rapid green purification and ultrasensitive simultaneous detection of urinary NPT, IXP and BPT for exploratory clinical studies. Download: Download high-res image (154KB) Download: Download full-size image