Use of multivariate statistical techniques to optimize the separation of 17 capsinoids by ultra performance liquid chromatography using different columns Janclei P. Coutinho a , Gerardo F. Barbero b,n , Oreto F. Avellán c , A. Garcés-Claver c , Helena T. Godoy a , Miguel Palma b , Carmelo G. Barroso b a Faculty of Food Engineering, University of Campinas, Monteiro Lobato Street, 80, 13083-862 Campina, SP, Brazil b Department of Analytical Chemistry, Faculty of Sciences, University of Cádiz, Agrifood Campus of International Excellence (CeiA3), PO Box 40, 11510 Puerto Real, Cádiz, Spain c Agrifood and Technology Centre of Aragón (CITA), Montañana Avenue, 930, 50059 Zaragoza, Spain article info Article history: Received 17 June 2014 Received in revised form 24 October 2014 Accepted 2 November 2014 Available online 20 November 2014 Keywords: Capsinoids Peppers UHPLC Multivariate statistical technique Derringer and Suich optimization abstract In this work a multivariate statistical tool (Derringer and Suich optimization) was proposed for the separation of seventeen capsinoids (natural and synthetic) using the UHPLC-DAD chromatography. Capsinoids were analyzed at 280 nm. The variables optimized were the mobile phase (water (0.1% acetic acid as solvent A) and acetonitrile (0.1% as solvent B)), gradient time and flow rate. Two columns with different length (50 and 100 mm) were used for the chromatographic separation. The two columns used properly separated the seventeen capsinoids, however the 100 mm column length showed a better chromatographic separation with a shorter run time and smaller peak widths. These results provided better values of limit of detection and quantification for the 100 mm column length. The better conditions of separation with the 100 mm column length were established with: initial mobile phase with 41.8% of solvent B; 3.96min of linear gradient time to reach 100% of solvent B; flow rate of 0.679 mL min À1 . A validation of the method has been done with excellent values of repeatability (RSD o1.92) and intermediate precision (RSD o3.92). The developed method has been applied to real samples. Capsiate has been identified and quantified in some varieties of peppers. & 2014 Elsevier B.V. All rights reserved. 1. Introduction Chilli peppers are the spicy fruits from plants of the genus Capsicum (Solanaceae), which are native to Central and South America [1]. Peppers are used as spices to add aroma and flavor to foods and they are commercially very important [2]. The main characteristic of peppers is their pungency, which is caused by chemical compounds known as capsaicinoids [3–5]. Apart from capsaicinoids, this fruit is also a source of other nutraceutical compounds such as phenolic compounds, carotenoids, ascorbic acid and tocopherol [6]. However, the consumption of these functional compounds is limited by the pungent flavor caused by capsaicinoids [7]. Capsinoids are non-pungent compounds that are similar to capsaicinoids in terms of structure and biological activities and they were recently isolated from some sweet peppers. Capsinoids have been shown to have similar benefits to capsaicinoids, e.g. anti-inflammatory, analgesic [8], anti-microbial, anti-mutagenic, anti-tumor [9] and antioxidant properties [10], but they do not have a pungent flavor [11–14]. The structural difference between the capsaicinoids and capsinoids is the way in which the carbon chain is bound to the aromatic ring: by an amide moiety in capsaicinoids and by an ester moiety in capsinoids [7]. Capsinoids have proven to be beneficial for human health since they promote the body’s metabolism and suppress body fat accumulation [15–18]. Quantification of the major and minor capsinoids in chilli peppers is problematical because the analysis of these compounds has not been studied previously. There are some reports on the synthesis of capsinoids and their biological activities have been studied [14,19–21]. Very few studies have been carried out on the separation of capsiate and dihydrocapsiate [7] and the chromato- graphic separation and quantification of minor compounds in chilli peppers have not been studied at all. The main benefit of the optimization process is the reduction of the time and costs of the general process. The multivariate Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/talanta Talanta http://dx.doi.org/10.1016/j.talanta.2014.11.004 0039-9140/& 2014 Elsevier B.V. All rights reserved. n Corresponding author. Tel.: þ34 956 016360; fax: þ34 956 016460. E-mail address: gerardo.fernandez@uca.es (G.F. Barbero). Talanta 134 (2015) 256–263