Journal of Chromatography A, 1074 (2005) 81–88 Development of a triple hyphenated HPLC–radical scavenging detection–DAD–SPE–NMR system for the rapid identification of antioxidants in complex plant extracts Audrius Pukalskas a,b , Teris A. van Beek a,∗ , Pieter de Waard a a Natural Products Chemistry Group, Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands b Department of Food Technology, Kaunas University of Technology, Radvilenu pl. 19, LT-50015 Kaunas, Lithuania Received 14 January 2005; received in revised form 10 March 2005; accepted 14 March 2005 Available online 12 April 2005 Abstract A rapid method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining an HPLC with on-line radical scavenging using DPPH • as a model radical and an HPLC–DAD–SPE–NMR system. Using this method a commercial rosemary extract was investigated. All major compounds present in the extract were collected on SPE cartridges after their separation. Advantages of on-line SPE peak trapping are the possibility to perform HPLC with non-deuterated solvents, a concentration effect and being able to record NMR spectra in pure 100% deuterated solvents. After comparing DAD and DPPH scavenging chromatograms, 1 H NMR spectra of compounds having radical scavenging activities were recorded. Afterwards all compounds were collected and infused into an ESI-MS. The five main active compounds – carnosol, carnosic acid carnosaldehyde, 12-methoxycarnosic acid and epiisorosmanol – could be identified from the combined UV, NMR and mass spectral data without actually isolating them. It was possible to record on-line an HMBC spectrum of carnosic acid. Also one compound was tentatively identified as epirosmanol methyl ether. © 2005 Published by Elsevier B.V. Keywords: Rosemary; Radical scavengers; On-line detection; Identification; LC–NMR 1. Introduction There is more and more evidence that oxidised lipids could have negative health implications, for instance in the development of coronary heart disease, carcinogenesis and even ageing [1]. To retard the oxidation process and prolong the shelf life of food containing (multiple) unsaturated fats, antioxidants are frequently added. Purified antioxidative extracts from rosemary leaves have achieved widespread commercial application as a food additive [2–13]. In some products rosemary extracts gave results similar to those of synthetic antioxidants like butylated hydroxytoluene (BHT) and butylated hydroxyanisole BHA [1]. Crude rosemary extracts possess a green colour and a rather strong odour. ∗ Corresponding author. Tel.: +31 317 482376; fax: +31 317 484914. E-mail address: teris.vanbeek@wur.nl (T.A. van Beek). Therefore, usually processed extracts are used which are more neutral with regard to colour, taste and smell. In order to avoid losing antioxidants during the processing, knowledge on the activity and identity of individual antioxidants is essential. Thus much work has been carried out on the chem- ical composition of rosemary extracts [14–21]. Compounds responsible for the antioxidant properties are rosmarinic acid and phenolic diterpenes, such as carnosol, carnosic acid, rosmanol, epirosmanol and isorosmanol [7,20]. Obtaining information on the antioxidant activity and identity of individual constituents in complex plant extracts, such as rosemary, is normally a time-consuming task. Each compound has to be purified to homogeneity and then its ac- tivity and structure can be determined with off-line methods. Recently a technique has become available to measure the radical scavenging activity of individual compounds on-line when they elute from an HPLC column [22–24]. As all of 0021-9673/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.chroma.2005.03.089