Determination of multiple risk substances in cultured meat biomass and washing solutions using LC-MS/MS: method development, validation, and risk assessment

Cultured meat presents significant potential as a sustainable supplement to traditional livestock farming. Addressing the critical challenge of residual chemical substances from the culture media, this study established a rapid method combining protein precipitation (PP) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of 12 risk substances in both porcine cultured meat biomass and washing solutions. All validation parameters, including linearity, sensitivity, precision, accuracy, recovery, matrix effect, dilution integrity and stability, met the predefined acceptance criteria. The results demonstrated that consecutive centrifugal washing cycles significantly reduced residue levels in porcine cultured meat biomass, with the majority of the 12 target substances effectively removed after four cycles. Analysis of 15 production batches revealed that 9 out of the 12 target substances were below the limit of detection (LOD) in the final product. For the remaining three substances, the mean concentrations were 307.91 μg/kg for forskolin, 568.04 μg/kg for thymidine, and 397.61 μg/kg for N -acetylcysteine. A target hazard quotient (THQ) assessment confirmed that none of the 12 residues posed a health risk. These findings provide the technical groundwork and theoretical rationale needed to optimize quality and safety specifications in the cultured meat industry.