Development and Validation of an LC-MS/MS Method for the Determination of Alternaria Mycotoxins in Hepatic Tissue

The presence of Alternaria mycotoxins in hepatic tissue of both human and animal origin remains unexplored. This work describes the development of an analytical method based on salt-assisted liquid-liquid extraction (SALLE) and ultrahigh-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) for the determination of six main Alternaria mycotoxins and related metabolites. Sample treatment was fully optimized, including sample mass, extraction solvent, and volume and sodium chloride mass. The method was validated, achieving calibration curve R² values above 0.99 and limits of detection between 0.01 and 1.46 µg kg^-1. Moreover, satisfactory trueness (apparent recoveries between 84% to 111%) and precision (RSD values below 10%) were achieved, complying with EU requirements. Matrix effects in terms of signal suppression/enhancement varied between 53% for TeA and 78% for AME. Applied to real liver samples (20 human and 20 animal), alternariol monomethyl ether (AME) was found in pig liver, while alternariol (AOH) and tentoxin (TEN) were found in human forensic liver tissues. No other Alternaria mycotoxin metabolites were detected. This methodology is the first validated approach for determining Alternaria mycotoxins in liver tissue.