Analytical, Nutritional and Clinical Methods Chemometrics for the resolution of co-eluting peaks of b- and c-tocopherols in RP-HPLC: Determination in edible oils and dietary supplements T. Galeano Dı ´az * , M.I. Rodrı ´guez Ca ´ceres, B. Rolda ´n Murillo * Department of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain Received 18 October 2006; received in revised form 14 February 2007; accepted 10 March 2007 Abstract This paper deals with the quantification of b- and c-tocopherol by RP-HPLC, using a C18 column as stationary phase, and MeOH:H 2 O (90:10, v/v) as mobile phase (flow rate, 2 mL min 1 ). Both analytes appear together in the same chromatographic peak and, as consequence, a partial least squares (type PLS-1) chemometric method has been developed to analyze them separately. Different calibration matrices have been done with the fluorescence spectra, excitation and emission, absorbance and derivative absorbance spec- tra, obtained across the peak or in the peak apex. The matrix of data constructed with the derivative absorbance spectra obtained in the peak apex is the one that gives best results. The method has been successfully applied to edible oils and dietary supplements. No pre- treatment of the samples was needed, only a dilution in 2-propanol was effected. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Tocopherols; RP-HPLC; PLS; Edible oil; Dietary supplements 1. Introduction Fat-soluble vitamins comprise vitamins A, D, E and K. Recommended dietary allowances (RDA) have been pub- lished all around the world. Some foods such as margarine, milk products, dietetic foods and infant formulas are com- monly fortified with vitamins A, D and E. The addition of vitamins to a particular processed food is intended to pro- vide a specific proportion of the RDA (Handbook of Food Analysis, 1996). General principles for the addition of nutrients to foods have been established by the Codex Ali- mentarius Commision (Codex Alimentarius Commission, 1987) and the US Food and Drug Administration (FDA, 1987). Vitamin assays in foods are carried out for several pur- poses, among them, to provide data for food composition and for quality assurance. Eight vitamers of vitamin E occur in nature: four tocopherols and four tocotrienols. Tocopherols are methyl-substituted derivatives. Tocophe- rols and tocotrienols are designated as a-, b-, c- and d- according to the number and position of the methyl groups in the chromanol ring. The role of vitamin E in the body can be explained in general as a lipid antioxi- dant in stabilizing subcellular membranes, but, it is worth noting that every vitamer varies widely in biological activity. The problem of the separation of a-, b-, c- and d-T has been the subject of numerous papers (Abidi, 2000; Pyka & Sliwiok, 2001; Rozzolo & Polesello, 1992). Abidi (2000) reviewed exhaustively the chromatographic analysis of tocol-derived lipid antioxidants performed until the date. The paper compiles published NP- and RP-HPLC and concluded that NP-HPLC have been used for the majority of investigators. It is worth noting that tocotrienols does not interfere in the analysis of tocopherols (Abidi & Ren- nick, 2001; Chen & Bergman, 2005). 0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.03.032 * Corresponding authors. Tel./fax: +34 924289375 (B. Rolda ´n Murillo). E-mail addresses: tgaleano@unex.es (T. Galeano Dı ´az), broldan@ unex.es (B. Rolda ´n Murillo). www.elsevier.com/locate/foodchem Food Chemistry 105 (2007) 1583–1590 Food Chemistry