Analytica Chimica Acta 514 (2004) 37–43 Sequential injection analysis-based flow system for the enzymatic determination of aspartame Rosa M. Peña a,∗ , José L.F.C. Lima b , M.Lúcia M.F.S. Saraiva b a Departamento de Qu´ ımica Anal´ ıtica, Nutrición y Bromatolog´ ıa, Facultad de Ciencias, Universidad de Santiago de Compostela, Alfonso X El Sabio s/n, 27002 Lugo, Spain b Requimte/Departamento de Qu´ ımica F´ ısica, Faculdade de Farmácia, Universidade do Porto, Rua An´ ıbal Cunha 164, 4050-047 Porto, Portugal Received 4 November 2003; received in revised form 2 March 2004; accepted 8 March 2004 Abstract The versatility of sequential injection analysis systems as a sampling handling approach combined with the flexibility of the individual solenoid valves was conjugated for the automatisation of the proposed new enzymatic method for the determination of aspartame in commercial sweetener tablets. The method involves the enzymatic conversion of aspartame in hydrogen peroxide by the -chymotrypsin–alcohol oxidase system, followed by the use of 2,2 ′ -azinobis(3-ethylbenzthiazolin-sulfonic acid-6) (ABTS) as electron donor for peroxidase. -Chymotrypsin and alcohol oxidase enzymes were immobilised on activated porous silica beads. No activity loss in the reactors was detected throughout 60 days. The calibration plots were linear up to 350 mg l -1 , with a detection limit of 2.16 mg l -1 of aspartame. Relative standard deviations (R.S.D.) of 3.0 and 2.4% (n = 10) for samples containing 25.8 and 51.9 mg l -1 of aspartame, respectively, were obtained. The method was applied to the determination of aspartame in commercial sweetener tablets; the results obtained were compared to those provided by a HPLC method and revealed no statistical differences for a 95% confidence level. © 2004 Elsevier B.V. All rights reserved. Keywords: Aspartame determination; SIA; Enzymatic method; Commercial sweetener tablets; -Chymotrypsin; Alcohol oxidase; Peroxidase 1. Introduction Aspartame (N-l--aspartyl-l-phenylalanine 1-methyl es- ter) is an artificial sweetener of low caloric value, which despite being organoleptically similar to sucrose is approx- imately 180 times sweeter [1]. Aspartame is currently per- mitted for food and beverage and/or tabletop sweetener use in more than 90 countries, which implies the development of analytical methodologies easily reproducible under nor- mal laboratory conditions, without the need for sophisticated equipment and specially trained personnel [2]. Several spectrophotometric procedures are described in the literature to determine aspartame, but these are either time consuming or do not have the selectivity required for its determination in some commercial samples [3–5]. Chro- matographic procedures [6–11] in spite of being perhaps the most commonly used for the determination of aspartame, ∗ Corresponding author. E-mail address: qarosa@lugo.usc.es (R.M. Peña). apart from the time and cost involved per analysis, they require extensive pre-treatment of the food sample prior to the chromatographic operation. Continuous flow pro- cedures present themselves as interesting alternatives for the automatisation of the different stages of an analytical process, as an increase in analytical efficiency is obtained. However, only some continuous flow procedures based on spectrophotometric detection or on the use of biosensors have been reported for the determination of aspartame. Nev- ertheless, the spectrophotometric methods use reagents that require either special precautions in handling and storage or need a water bath at 60 ◦ C [12–14]. The enzyme based biosensor procedures had the drawback of electrode stabil- ity, which decreased after around 250 assays [15–17]. The use of flow methodologies for the determination of aspar- tame with spectrophotometric detection that take advantage of the selectivity and specificity of the use of enzymes, has never been carried out. Some enzymatic batch methods [18,19] for its determination are described in the literature, but are slow procedures that involve an extensive incubation period. 0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.03.015