Simultaneous determination of 12 lipophilic shellfish toxins in human plasma, serum, and urine using UPLC-MS/MS combined with PRiME HLB μElution platform.

Lipophilic shellfish toxins (LSTs) pose significant health risks through contaminated seafood consumption, necessitating robust monitoring methods, which is critical for early and definitive diagnosis. However, trace analysis of LSTs in human biological fluids remains challenging due to complex matrices and ultra-low concentrations. For this purpose, a rapid, sensitive, and high-throughput UPLC-MS/MS method was developed and validated for the simultaneous determination of 12 LSTs in human plasma, serum, and urine using a μElution 96-well plate for sample preparation. By comparing the extraction efficiency of different types of plate, optimizing dilution factors of eluates, and evaluating matrix effects, a workflow based on the PRiME HLB μElution plate was established for extraction and cleanup, effectively minimizing matrix interference and stabilizing recovery. Chromatographic separation was achieved on a C18 column within 8 minutes using a gradient of water and acetonitrile (both containing 6.7 mmol/L ammonium hydroxide). Target toxins were monitored via multiple reaction monitoring mode with polarity switching (±), and quantified using matrix-matched calibration curves. The method was validated over a linearity of 0.20-30 μg/L (R² > 0.995), with LOQs between 0.03 and 0.77 μg/L. Average recoveries ranged from 69.0 % to 124.0 % with RSDs of 1.1 %-15.3 %. The μElution procedure surpassed conventional SPE in terms of recovery and robustness across all matrices. The method was successfully applied to the analysis of samples from healthy volunteers and LST-dosed rats, detecting two toxins in rat urine. Its simplicity, speed, sensitivity and high-throughput make it a robust tool for LST intoxication detection and biomonitoring.