The paper reviews the methods described in the literature for the determination of total dissolved free primary amines (TDFPA) by fluorescence. A wide set of reaction conditions can be found, but they rely on few experiments for their validation. Among fluorogenic compounds, o-plithaldialdehyde (OPA) is more sensitive than fluorescamine and was thus examined here. However, the use of mercaptoethanol (ME) in the reaction (as ail additional derivatization compound) is able to generate unreliable results, in particular when standardization relies on glycine. We suggest replacing ME with 3-mercaptopropionic acid (MPA) which induces more stable and comparable fluorescence among amine Compounds. A systematic study was therefore undertaken to define reagent concentrations and pH effects on the reaction rates for a variety of primary amines with particular focus on amino acids. The reaction rate is increased by increasing OPA concentration and pH, but slowed by excess MPA. Ammonium interference is influenced by several factors, but spectral investigation showed that the choice of conditions can drastically reduce it. The magnitudes of natural and OPA-induced background fluorescence signals have been assessed in various mediums and it is shown that their contribution to the signal amounts to a large fraction, when not most, of the measured fluorescence. A segmented flow method is proposed with a protocol for adequate correction of biases. (c) 2005 Elsevier B.V. All rights reserved.