Rapid Determination of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Human Urine Based on Polystyrene/Graphene Oxide Packed-Fibers Solid-Phase Extraction Coupled With High-Performance Liquid Chromatography and Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry

Vitamin D plays a crucial role in skeletal metabolism and is implicated in various diseases. Several studies have indicated that human vitamin D status can be evaluated through urine hydroxyvitamin D levels, in addition to plasma hydroxyvitamin D concentrations. In this study, we developed a novel method for the detection of trace amounts of 25-hydroxyvitamin D₃ [25(OH)D₃] and 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] in urine. This method is based on high-performance liquid chromatography coupled with atmospheric pressure chemical ion source-tandem mass spectrometry, integrated with packed-fibers solid-phase extraction. The composite polystyrene/graphene oxide (PS/GO) nanofibers were synthesized as the sorbent for concentrating 25(OH)D₃ and 24,25(OH)₂D₃, achieving a twentyfold increase in sensitivity when analyzing human urine samples. Fourier-transform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy mapping analysis were employed to characterize GO, PS, and PS/GO. The results indicated that the composite nanofibers were successfully synthesized. In addition, we investigated the relationship between the extraction performance of the fibers and the electrospinning process at varying flow rates of spinning solution through morphological studies and Brunauer-Emmett-Teller surface area analysis. During the extraction process, purification, concentration, and desorption were accomplished within a single step. The established method demonstrated excellent sensitivity and efficiency. Under optimal conditions, limits of detection (signal-to-noise ratio = 3) were found to be 0.33 ng/mL for 25(OH)D₃ and 0.19 ng/mL for 24,25(OH)₂D₃; furthermore, linearity was deemed acceptable across urine samples. Recovery rates ranged from 89.5% to 109.7% for 25(OH)D₃ and from 90.3% to 103.1% for 24,25(OH)₂D₃, respectively.

LC‐MS/MS; nanofibers; solid‐phase extraction; urine; vitamin D3 metabolites.