The use of algae as a foodstuff is rapidly expanding worldwide from the East Asian countries, where they are also used for medical care. Harmala alkaloids (HAlk) are a family of bioactive compounds found in the extracts of some plants, including wakame (Undaria pinnatifida), an edible marine invasive algae. HAlks are based on a characteristic β- carboline structure with at least one amino ionizable group. In this work, we report the successful separation of a mixture of six HAlks (harmine, harmaline, harmol, harmalol, harmane, and norharmane) by capillary electrophoresis ion-trap mass spectrometry (CE-IT-MS) in less than 8 min. Optimum sepa- ration in fused-silica capillaries and detection sensitivity in positive-ion mode were achieved using a background electro- lyte (BGE) with 25 mmol L−1 ammonium acetate (pH 7.8) and 10 % (v/v) methanol, and a sheath liquid with 60:40 (v/v) isopropanol–water and 0.05 % (v/v) formic acid. The separa- tion method was validated in terms of linearity, limits of de- tection and quantification, repeatability, and reproducibility. Later, a sample pretreatment was carefully optimized to Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8579-4) contains supplementary material, which is available to authorized users. M. Tascon : L. G. Gagliardi (*) Laboratorio de Investigación y Desarrollo de Métodos Analíticos (LIDMA), Universidad Nacional de La Plata, Calle 47 y 115, 1900 La Plata, Buenos Aires, Argentina e-mail: leogagliardi@quimica.unlp.edu.ar L. G. Gagliardi Centro de Investigación y Desarrollo en Tecnología de Pinturas (CIDEPINT-CIC-CONICET), Av. 52 y 120, 1900 La Plata, Buenos Aires, Argentina F. Benavente : V. M. Sanz-Nebot Departamento de Química Analítica, Nutrition and Food Safety Research Institute (INSA-UB), Universidad de Barcelona, Av. Diagonal 645, 08028 Barcelona, España determine HAlks in commercial wakame samples with excel- lent recovery and repeatability. For the complex wakame ex- tracts, the MS–MS fragmentation patterns of the different HAlks were useful to ensure a reliable identification. The complete procedure was validated using the standard- addition calibration method, determining matrix effects on the studied compounds. Harmalol, harmine, and harmaline were naturally present in the samples and were quantified at very low concentrations, ranging from 7 to 24 μg kg−1 dry algae. Keywords Capillary electrophoresis . Mass spectrometry . MS–MS . Undaria pinnatifida . Harmala alkaloids . Validation