An automatic online solid-phase dehydrate extraction-ultra-high performance supercritical fluid chromatography-tandem mass spectrometry system using a dilution strategy for the screening of doping agents in human urine.

An automatic online solid-phase dehydrate extraction (SPDE)-ultra-high performance supercritical fluid chromatography (UHPSFC)-MS/MS system was developed in this study, in which the automatic SPDE procedure was coupled with UHPSFC to allow UHPSFC to analyze aqueous samples directly. Moreover, a pre-column dilution strategy was employed, which focused the analytes in strong desorption solvent on the column head and helped to obtain narrow and symmetric peaks. The online SPDE-UHPSFC-MS/MS system was firstly applied to the screening of 45 prohibited substances in human urine for doping control, during which all the mechanisms and features of the online system were fully studied. The majority (91%) of the target compounds achieved weak matrix effects (80-120%), indicating that the online method was accurate and reliable thanks to the SPDE procedure and efficient UHPSFC separation. Owing to the reduction of the matrix effects, large volume injection and the pre-column dilution, the online system could achieve high sensitivity with the LODs ranging from 0.0380 ng L to 1.24 μg L. Under the optimized conditions, the extraction recoveries of 66% target analytes were more than 50%. All the target compounds showed good linearity with linear correlation coefficients higher than 0.9928. The accuracy values of all the spiked prohibited substances were within 80.8-119.7%, while the RSDs% for the intra-/inter-day precision were within 10.8% and 15.4%. Compared with the dilute-and-shoot-ultra-high performance liquid chromatography-MS/MS method, in which the urine samples were simply diluted before analyzing, this online method was superior in sensitivity and reducing matrix effects, which demonstrated its utility in doping control. Compared with the previously reported online SPE-SFC system, the online SPDE-UHPSFC-MS/MS system showed advantages in automation, efficiency, sensitivity and chromatographic performance. In summary, the online SPDE-UHPSFC-MS/MS system is capable of analyzing complex aqueous samples.