Development and application of automatic magnetic solid phase extraction assisted High-resolution mass spectrometry...

Over the past three decades, numerous deaths caused by the illicit use of fentanyl analogues have drawn worldwide concern. However, the detection of trace levels of fentanyl compounds remains challenging due to labor-intensive sample pretreatment, low sensitivity, and limited screening efficiency. In this study, we developed and validated a rapid and sensitive method based on high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of 26 fentanyl analogues and metabolites in urine samples. After automated magnetic solid-phase extraction (auto-MSPE), analytes were separated on a C18 column and detected using MRM HR mode. The limits of detection (LOD) and limits of quantification (LOQ) ranged from 0.01 to 0.05 ng/mL and 0.04 to 0.1 ng/mL, respectively. This approach required only 30 min for the pretreatment of 32 urine samples, meeting routine detection requirements while achieving a green and economical workflow. Within the concentration range of 0.1-100 ng/mL (0.5-100 ng/mL for two compounds), all analytes exhibited good linearity with correlation coefficients (R² > 0.99). Accuracy ranged from 75.1 % to 117.0 %, extraction recoveries from 72.1 % to 100 %, and matrix effects from 64.2 % to 111.7 %. The method was successfully applied to the detection and quantification of fentanyl and its major metabolite norfentanyl in authentic urine samples. These results demonstrate the potential of this method for the reliable detection of fentanyl analogues in forensic and clinical toxicology.