Analytical Methods Determination of lipid and protein hydroperoxides using the fluorescent probe diphenyl-1-pyrenylphosphine Ricard Bou a,b, * , Bingcan Chen a , Francesc Guardiola b , Rafael Codony b , Eric A. Decker a a University of Massachusetts, Department of Food Science, Amherst, MA, USA b University of Barcelona, Nutrition and Food Science Department, XaRTA-INSA, Barcelona, Spain article info Article history: Received 2 September 2009 Received in revised form 25 January 2010 Accepted 1 May 2010 Keywords: Protein hydroperoxide Lipid hydroperoxide Diphenyl-1-pyrenylphosphine DPPP Method validation abstract By means of two alternative methods lipid and protein hydroperoxides (HP) were determined by fluo- rometry using the diphenyl-1-pyrenylphosphine (DPPP) probe. It has been shown that the formation of the fluorescence was influenced by the type of solvent and HP whereas the presence in the media of antioxidants such tocopherol and butylated hydroxytoluene had no effect. The combination of the chloroform:methanol (2:1, v/v) solvent mixture that is widely used for lipid extraction was combined with suitable solvents to develop a method with the maximum performance in determining HP in lipid extracts. Using a variety of lipids and lipid extracts, the final method proposed agreed well with the thio- cyanate method for HP determination. In addition, the DPPP method was very sensitive, precise, accurate, free of interferences and specific for the determination of lipid soluble HP. DPPP can be also used to mea- sure HP soluble in hydroalcoholic media. This alternative procedure showed a similar performance to its lipid soluble equivalent and was able to measure hydrogen peroxide promoted peroxidation of bovine serum albumin and water soluble HP in protein extracts. With the addition of triphenylphosphine the hydroalcoholic method is specific for the determination of protein HP. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Organic hydroperoxides (HP) are oxidative mediators and prod- ucts of peroxidation chain reactions produced by free radicals and enzymes such as lipoxygenase. They can be originated from the oxidation of unsaturated fatty acids and other lipid classes (Gutter- idge, 1995) but also from proteins and amino acids (Gebicki, 1997; Gebicki & Gebicki, 1999). Lipid HP are odourless but their formation and breakdown in foods lead to loss of nutrients with compounds with negative bio- logical effects and off-flavours also being formed (Diplock et al., 1998; Esterbauer, Schaur, & Zollner, 1991; Nawar, 1996). There- fore, the food industry is interested in both inhibiting peroxidation and determining their concentration which gives an indication of the oxidative status, especially in a relatively non-oxidised prod- uct. In biological matrices, HP measurement, in connection with free radicals and other reactive oxygen species, has been used as indication of oxidative stress (Halliwell & Chirico, 1993; Mehrotra, Ling, Bekele, Gerbino, & Earle, 2001; Sivaram, Suresh, & Indira, 2003) which is associated with various diseases (Casetta, Govoni, & Granieri, 2005; Castro & Freeman, 2001; Esterbauer, Wag, & Puhl, 1993; Finkel, 1998) and ageing (Holbrook & Ikeyama, 2002; Stadtman & Berlett, 1997). Until recently, proteins were not held to be biologically signifi- cant targets for reactive oxygen species but research has shown that protein form HP and can be responsible of DNA cross-linking (Gebicki & Gebicki, 1999). Mildly oxidised proteins are readily de- graded and removed from the cell but when proteins are highly oxidised they aggregate and/or their solubility decreases which makes their elimination difficult (Cecarini et al., 2007; Grune, Merker, Sandig, & Davies, 2003). These highly oxidised proteins may contribute to several neurodegenerative diseases such as Alzheimer and may be involved in the ageing process (Grune et al., 2003; Stadtman, 2006; Widmer, Ziaja, & Grune, 2006). There- fore, highly oxidised proteins and protein HP can be useful markers of oxidative stress but there is a lack of simple and sensitive methods to determine these compounds. Although various methods have been proposed to measure HP, the instability and diversity of HP in complex foods or biological matrices make it difficult to conduct accurate, sensitive and simple analysis. Chromatographic (GC and HPLC) and spectroscopic (NMR and ESR) methods showed high sensitivity, selectivity and repro- ducibility (Dobarganes & Velasco, 2002; Frankel, Neff, & Weisleder, 1990; Hughes, Smith, Horning, & Mitchell, 1983; Yamamoto, 1994; Yang et al., 1991) but not all laboratories have the necessary instru- mentation so their application to routine analysis is compromised. 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.05.003 * Corresponding author at: University of Barcelona, Nutrition and Food Science Department, XaRTA-INSA, Barcelona, Spain. E-mail address: ricard_bou@ub.edu (R. Bou). Food Chemistry 123 (2010) 892–900 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem