High-throughput 96-well solid-phase extraction for preparation of tetracycline followed by liquid chromatography and mass spectrometry analysis.

Tetracyclines abuse has frequently occurred in aquaculture against bacteria, rickettsiae, spirochetes, and mycoplasmas. In this study, a high-throughput sample preparation method was developed using 96-well plate solid-phase extraction (p-SPE) and the extract was analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The experimental conditions were optimized such that the pH is 4, the eluting solvent is methanol (2 mL), and the sorbent is hydrophilic-lipophilic balance (HLB) microsphere. The whole protocol was validated, and it showed that the tetracyclines were linear with correlation coefficients ≥ 0.9990, precision and accuracy (RSD%) in 3.9-6.1%, and mean recoveries of 88.6-103.6%. To exhibit the potential of 96-well p-SPE as a routine tool for inspection and quarantine, fresh aquatic samples were tested, and among which positive samples were observed. This method was demonstrated to be promising for the purification and enrichment of tetracyclines with reduced time and labor, and indeed practically and particularly suitable for widespread tetracyclines analysis.