96-Sample Parallel BioSPME-DART-MS Platform for Rapid Screening of Fentanyl and its Analogues in Biofluids

Abstract Solid-phase microextraction (SPME) with biocompatible coating material is highly compatible with ambient mass spectrometry (AMS), such as direct analysis in real time (DART), for improving sensitivity and reducing matrix effects. However, the practical throughput of SPME−DART-MS remains limited by the mismatch between parallel sample preparation and device-to-source transfer. Here, we introduce a 96-sample parallel BioSPME-DART-MS platform that integrates automated SPME extraction, 96-channel liquid transfer, and a matched 96-position DART mesh interface. This design enables 96 biological samples to be processed in a plate-based format, greatly improves workflow transferability. Thin HLB/PAN-coated SPME blades were used to provide bio-compatible extraction and cleanup from whole blood, plasma, and urine. After desorption in 200 μL of solvent, only 5 μL of the extract was transferred to the DART mesh for rapid MS/MS screening, while the remaining solution was retained for sequential LC−MS/MS confirmation without additional sample preparation. The system enabling an average analysis time of approximately 40 s per sample. Using six fentanyl and its analogues as model analytes, the BioSPME-DART-MS method achieved good sensitivity across the three biofluids, with limits of detection of 0.10−0.50 ng mL−1 in whole blood, 0.03−0.20 ng mL−1 in plasma, and 0.01−0.20 ng mL−1 in urine. In addition, a comparative study of absolute matrix effects revealed that chromatography-free DART-MS and blade spray-MS remained susceptible to ion suppression, whereas LC−ESI-MS showed negligible matrix effects. This platform establishes a high-throughput, two-tier SPME−MS workflow that combines rapid ambient MS screening with chromatography-based confirmation.