Rapid screening method of glucocorticoid residues in sample-limited cell-cultured meat

The emergence of cell-cultured meat as a sustainable protein source necessitates robust safety assessment frameworks, particularly for novel inputs like glucocorticoids-potent media supplements used to enhance cell growth. This study addresses the critical analytical gap for monitoring these compounds in complex cultured meat matrices. A sensitive and selective isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry method was established for 40 glucocorticoids, overcoming key challenges including significant matrix effects and the separation of six critical isomer pairs. Through optimized miniaturized sample preparation (0.2 g) combined with chromatographic separation on a BEH Phenyl column, the method achieved quantification limits (MQLs) of 0.5-3 μg/kg in cell-cultured biomass and supernatant, with recoveries of 70-118%. The method was applied to analyze residual glucocorticoids during the cell-cultured pork production. Hydrocortisone was the only glucocorticoid detected, with a concentration of 3.15 ± 0.07 μg/kg in final biomass, well below the maximum residue limit for milk (10 μg/kg). Furthermore, a safety-assessed maximum intake (SAMI) limit was derived to guide future media formulation. This work provides the first systematic study of glucocorticoid residues in cell-cultured meat production, establishing a vital analytical method to support the safe development of the cell-cultured meat industry.