A new method based on matrix solid-phase dispersion (MSPD) on-line with a solid-phase extraction (SPE) cleanup process followed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) is presented for the determination of 3 macrocyclic lactone mycotoxins in fish tissues: zearalenone, alpha-zearalenol, and beta-zearalenol. The sample was prepared in a device that used a reversed-phase material (C18) or a normal-phase material (neutral alumina) as a matrix dispersing agent, and a graphitized carbon black cartridge was used for sequential cleanup by SPE. LC/MS/MS was used for selective determination. Isocratic elution with acetonitrile-methanol-water was used for LC separation; for MS/MS, 2 types of interfaces (a pneumatically assisted electrospray ionization interface or an atmospheric pressure chemical ionization interface) were evaluated and compared in terms of the intensity of the total ion current produced by each analyte. The use of highly selective MSPD on-line with SPE for sample preparation before analysis allowed the removal of interfering matrix compounds present in tissue extracts that would otherwise cause severe ionization suppression of zearalenone and its metabolites during the ionization process. Average recoveries at 100 ng/g were between 83 and 103% with C18 and > or = 67% with neutral alumina; the relative standard deviations were < 11% with C18 and < 18% with alumina. The limits of detection ranged from 0.1 to 1.0 ng/g. Sample preparation is simple to perform, no special technical equipment is required, and solvent volumes are minimal.