AbstractA reliable and sensitive method for determination of hepatotoxic microcystins in complicated matrices by frit‐fast atom bombardment liquid chromatography/mass spectrometry (Frit‐FAB LC/MS) is described. Immonium ions of constituent amino acids, which were obtained together with molecular ion species by FAB mass spectral analysis of standard microcystins RR, YR, LR, and [D‐Asp3] and [Dha7]microcystins LR using flow injection system composed of Frit‐FAB probe, showed potential for reliable identification of microcystins by Frit‐FAB LC/MS. Frit‐FAB LC/MS using a microbore column provided not only the baseline separation of standard microcystins RR, YR, and LR but 200‐fold higher sensitivity than that using conventional column. Furthermore, when a selected ion monitoring (SIM) technique was used, the detection limits of microcystins RR, YR, and LR were 300, 350, and 400 pg, respectively, at a signal‐to‐noise ratio of 5:1, and calibration curves of each microcystin showed a linear relationship from 2 ng to 50 ng. Finally, identification and quantitative analyses of microcystins in water samples were carried out. © 19