Flow-injection fluorimetric assay of nitrogen-containing substrates by on-line enzymatic generation of ammonia

A fluorimetric flow-injection method for the determination of nitrogen-containing substrates, which can be enzymatically degraded to ammonium/ammonia is described. The generated ammonia is detected fluorimetrically after on-line derivatization with o-phthaldialdehyde (OPA) and sulphite. The derivatization procedure is optimized by a fractional factorial design at two levels and by a super modified simplex procedure to obtain a high sensitivity and a low detection limit for ammonia in the micromolar concentration range. Under the given flow conditions the detection limit is 1 μM, and considerable selectivity for ammonia over primary amines such as unconverted substrate and amino acids is achieved. Two enzymatic systems, incorporating immobilized creatinine iminohydrolase (CIH) and L-aspartase column reactors, respectively, are tested as model systems. The feasibility of the CIH system for the practical assay of creatinine in serum samples is demonstrated.