Fully automated robotic sample preparation coupled with UPLC-MS/MS for high-throughput screening of 19 nitazene...

An automated protein precipitation (PPT) method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed using an in-house developed automatic sample pretreatment platform. The platform integrates six sets of 8-position tube racks, a centrifuge vials rack, a reagent rack, an automatic decapper, a vortex mixer, a centrifuge, and an X-Y-Z axis robotic arm. The online sample preparation workflows and chromatographic conditions were systematically optimized for the comprehensive analysis of 19 nitazene analogues in human urine. The robotic platform operates in a pipeline-parallel processing mode, in which UPLC-MS/MS analysis (18 min per sample) is performed for one batch, while the subsequent batch of eight samples undergoes automated pretreatment (30 min per batch) without manual intervention. This design achieves a total turnaround time of 14.9 h for 48 samples, corresponding to an average processing time of 18.6 min per sample. Furthermore, the entire pretreatment procedure consumes only 400 μL of methanol per sample, thereby reducing organic solvent consumption. The method demonstrated good precision, with intra-day relative standard deviations (RSDs) of 1.2-7.0% and inter-day RSDs of 0.6-5.1%, indicating satisfactory analytical reproducibility. Application of the method to 24 authentic urine samples identified 15 positives specimens, with N,N-dimethylamino etonitazene (0.4-168.5 ng/mL) being the most frequently detected analyte, together with three other nitazene analogues or metabolites. Overall, the developed automated PPT-UPLC-MS/MS platform provides a rapid, reproducible, and solvent-efficient approach for high-throughput screening of nitazene analogues in human urine, with potential applications in forensic toxicology and public health surveillance.