Analytica Chimica Acta 558 (2006) 310–318 Automatic method for determination of total antioxidant capacity using 2,2-diphenyl-1-picrylhydrazyl assay Lu´ ıs M. Magalh˜ aes, Marcela A. Segundo ∗ , Salette Reis, Jos´ e L.F.C. Lima REQUIMTE, Servi¸ co de Qu´ ımica-F´ ısica, Faculdade de Farm´ acia, Universidade do Porto, Rua An´ ıbal Cunha, 164, 4099-030 Porto, Portugal Received 10 October 2005; received in revised form 3 November 2005; accepted 4 November 2005 Available online 19 December 2005 Abstract In the present work, an automatic method based on multi-syringe flow injection analysis (MSFIA) was developed for the determination of total antioxidant capacity, measured as the cumulative capacity of the compounds present in the sample to scavenge free radicals, using the 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) reaction. The determination is based on the colour disappearance due to the scavenging of DPPH • by antioxidant compounds monitored spectrophotometrically at 517 nm. The influence of initial DPPH • concentration and sample dilution in the present methodology was studied. It was verified that the amount of DPPH • consumed by antioxidant standards (ascorbic and caffeic acids) was independent of the initial concentration of radical except for situations where DPPH • /antioxidant molar ratio was lower than the stoichiometric value. Furthermore, the sample dilution factor plays an important role for achieving results comparable to those from end-point batch method since the exhausting of scavenging ability of the sample should take place during the period of absorbance measurement. The proposed method was applied to several food products and the total antioxidant capacity was expressed as Vitamin C equivalent antioxidant capacity (VCEAC). The results obtained by the proposed method ranged from 1.1 to 318 mg of ascorbic acid/100 ml and they were statistically comparable to those provided by the batch method. The detection limit was 0.34 mg of ascorbic acid/100 ml and the determination frequency was about 13 h -1 with an excellent repeatability (R.S.D. < 1%, n = 10). © 2005 Elsevier B.V. All rights reserved. Keywords: Total antioxidant capacity; DPPH • ; Multi-syringe flow injection; Beverages 1. Introduction There is recent evidence that free radicals induce oxida- tive damage to biomolecules. This damage has been implicated in ageing and in several human pathologies such as cancer, atherosclerosis, rheumatoid arthritis and other diseases [1]. Fur- thermore, there is a considerable amount of studies indicating that the active dietary constituents of fresh fruit, vegetables and beverages, prevent these free radical-induced diseases and pro- tect against foodstuff oxidative deterioration [2–4]. These pro- tective effects have been attributed, in large part, to the antioxi- dants species (Vitamins C and E, carotenoids and polyphenolic compounds) which scavenge free radicals [5,6]. ∗ Corresponding author. Tel.: +351 22 2078994; fax: +351 22 2004427. E-mail address: msegundo@mail.ff.up.pt (M.A. Segundo). Several methodologies, based on free radical capture or formation suppression, are used to measure the antioxidant capacity of biological material and model compounds [7,8]. The most commonly used for their ease, speed and sensitivity are those involving chromogen compounds of a radical nature to simulate radical oxygen and nitrogen species. The most widely used assays are based on the scavenging of radical cation 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS •+ assay) [9] or of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH • assay) [10,11]. The presence of antioxidant species leads to the disappearance of these radical chromogens which can be followed by spectrophotometric methods. Recently, the DPPH • assay was implemented using automatic methods based on flow injection analysis (FIA) [12,13], sequen- tial injection analysis (SIA) [14] or HPLC-FIA [15,16], that were applied for screening and evaluation of scavenging capacity of several pure compounds and complex matrices such as plant extracts and beverages. In the HPLC-FIA method, the separated 0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.11.013