A novel automated micro solid phase extraction (µSPE) method to quantify 21 per- and polyfluoroalkyl substances compounds in biological samples

A semi-automated micro solid-phase extraction (μSPE) method has been developed and optimized for the quantification of 21 per- and polyfluoroalkyl substances (PFAS) across various biological matrices, including plasma, serum, urine, and liver tissue. This method employed μSPE cartridges integrated with a PAL-RTC robotic system, which enabled precise flow control and high-throughput procedures while ensuring exceptional extraction efficiency and reproducibility. Sample homogenization of liver tissues was achieved by bead-mill technology which eliminated sample cross-contamination and significantly reduced processing time. The extraction efficiency was influenced by the concentration of methanol in the sample, especially for longer-chain PFAS (C9–14). Extraction efficiencies of over 90% extraction were achieved with loading solutions containing 50–60% methanol in plasma and serum, and up to 80% methanol in water samples. The μSPE protocol included two sequential wash steps that provided excellent matrix clean-up, and reduced background signals by over 95% compared to traditional protein precipitation (PP) methods. The µSPE-LC-MS/MS method offers high sensitivity (method detection limits between 0.008 ng/mL and 0.048 ng/mL), along with good recoveries (90.3 to 105.1%), and precision (RSD