Journal of Chromatography A, 1233 (2012) 8–15 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A j our na l ho me p ag e: www.elsevier.com/locate/chroma Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries Raimondas Raudonis ∗ , Lina Raudone 1 , Valdas Jakstas 2 , Valdimaras Janulis 3 Department of Pharmacognosy, Lithuanian University of Health Sciences, Mickeviciaus Str. 9, LT-44307, Lithuania a r t i c l e i n f o Article history: Received 3 November 2011 Received in revised form 7 February 2012 Accepted 8 February 2012 Available online 15 February 2012 Keywords: HPLC post-column ABTS FRAP Antioxidant activity Fragaria L. a b s t r a c t ABTS and FRAP post-column techniques evaluate the antioxidant characteristics of HPLC separated compounds with specific reagents. ABTS characterize their ability to scavenge free radicals by electron- donating antioxidants, resulting in the absorbance decrease of the chromophoric radical. FRAP – is based on the reduction of Fe(III)–tripyridyltriazine complex to Fe(II)–tripyridyltriazine at low pH by electron- donating antioxidants, resulting in an absorbance increase. Both post-column assays were evaluated and compared according to the following validation parameters: specificity, precision, limit of detection (LoD), limit of quantitation (LoQ) and linearity. ABTS and FRAP post-column assays were specific, repeat- able and sensitive and thus can be used for the evaluation of antioxidant active compounds. Antioxidant active compounds were quantified according to TEAC for each assay and ABTS/FRAP ratio was derived. No previous records of antioxidative activity of leaves and fruits of strawberries (Fragaria viridis, Fra- garia moschata) research have been found. The research results confirm the reliability of ABTS and FRAP post-column assays for screening of antioxidants in complex mixtures and the determination of radical scavenging and ferric reducing ability by their TEAC values. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Oxidative stress plays a pivotal role in pathogenesis of cardio- vascular, neurodegenerative diseases, cancer and aging [1]. Antioxi- dants reduce oxidative stress by various mechanisms [2]. Research of natural antioxidants has increased in area of functional foods, agriculture and disease prevention [3–5]. Numerous assays with different mode of action have been established for the assessment of antioxidant activity [3,6,7]. Spectrophotometric studies evaluat- ing the total antioxidant capacity are convenient and easy adapt- able. These assays depend on single electron transfer or hydrogen atom transfer [8]. Most popular assays, based on single electron transfer, evaluate radical scavenging abilities (ABTS – 2,2-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid), DPPH – 2,2-diphenyl-1- picrylhydrazyl) and potential of ferric or cupric reducing (FRAP – ferric reducing antioxidant power, CUPRAC – cupric reducing antioxidant capacity) capacity expressed as Trolox equivalents [3,9–12]. Yet, they evaluate the additive and synergistic interre- lational impact of all compounds present in the sample as herbal ∗ Corresponding author. Tel.: +370 67843678. E-mail addresses: raimondas.raudonis@gmail.com (R. Raudonis), raudone.lina@gmail.com (L. Raudone), valdas.jakstas@med.kmu.lt (V. Jakstas), farmakog@kmu.lt (V. Janulis). 1 Tel.: +370 68241377. 2 Tel.: +370 67200844. 3 Tel.: +370 69802919. extracts contain a body of polyphenolic compounds with differ- ent structure and activity [13]. To elucidate the activity of separate compounds in complex extracts is not possible. Structure–activity relationships well established in numerous studies confirmed different modes of action of separate polyphenolic compounds [14,15]. It is purposeful to evaluate individual polyphenolic com- pounds in herbal extracts with antioxidant activity. Recently a body of researches has established on-line post- column methods for screening of antioxidants in complex mixtures [16–21]. HPLC separation is coupled with rapid identification of antioxidative active compounds. The pivotal advantages of these post-column reaction methods are that the antioxidant activity of an individual compound can be measured and its contribution to the total activity of a complex mixture can be estimated, and also the activity of an individual compound can be compared to other constituents in the mixture and their structure–activity rela- tionships can be determined [21,22]. These methods exclude the interactional effects of compounds as the detection occurs with separated analytes. Most of post-column assays use DPPH and ABTS radicals [16,21,23], while FRAP post-column assay, to the best of our knowledge, has not been installed previously. The latter assays can be performed in aqueous medium and low pH. As the assays are installed in the same conditions, it becomes possible to compare the radical scavenging and reducing abilities of antioxidant active compounds. Leaves and fruits of strawberries (Fragaria L. species) have long been used for medicinal and nutritional purposes [24]. The herbal 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2012.02.019