Analytical Methods Riboflavin and lumichrome in Dalmatian sage honey and other unifloral honeys determined by LC–DAD technique Carlo Ignazio Giovanni Tuberoso a,⇑ , Igor Jerkovic´ b , Ersilia Bifulco a , Zvonimir Marijanovic c , Francesca Congiu a , Dragan Bubalo d a Dipartimento Farmaco Chimico Tecnologico, University of Cagliari, via Ospedale 72, 09124 Cagliari, Italy b Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split, N. Tesle 10/V, 21000 Split, Croatia c Department of Food Technology, Marko Marulic ´ Polytechnic in Knin, Petra Krešimira IV 30, 22300 Knin, Croatia d Faculty of Agriculture, University of Zagreb, Svetošimunska 25, 10000 Zagreb, Croatia article info Article history: Received 14 September 2011 Received in revised form 16 April 2012 Accepted 26 June 2012 Available online 4 July 2012 Keywords: Riboflavin Umichrome LC–DAD Salvia officinalis L. honey abstract Riboflavin (vitamin B 2 ) and its metabolite lumichrome were quantified in 117 samples from 11 unifloral honeys types (Arbutus unedo L., Asphodelus microcarpus Salzm. et Viv., Citrus spp., Eucalyptus spp., Hedysa- rum coronarium L., Castanea sativa L. honeydew, Mentha spp., Paliurus spina-christi., Salix spp., Salvia offi- cinalis L., Satureja spp.). The quantification of these two compounds was performed by LC–DAD method which does not require sample purification. The proposed method in our study has low limits of detection and quantification, very good linearity in a large concentration range and very good precision. It allows simultaneous determination of 5-hydroxymethylfurfural (HMF) and known chemical biomarkers of unifl- oral honeys such as abscisic acid diastereomers, homogentisic acid, methyl syringate and kynurenic acid. No statistical correlation was observed between riboflavin and lumichrome content. Although, the con- centration of vitamin B 2 in honey may be too low (<6.1 mg/kg) to generate interest in the field of nutri- tion, the presence of its main metabolite lumichrome may be useful to determine the botanical origin of certain unifloral honeys. In fact, the analysis of 11 unifloral honey types showed that Dalmatian sage ( S. officinalis L.) honey is characterised by unusual high levels of lumichrome (20.2 ± 2.6 mg/kg). The botanical origin of lumichrome from sage flower was assessed by analysing bee-stomach extracts. Other analytical parameters, such as total phenols, antioxidant and antiradical activities, HMF and diastase activity were studied in Dalmatian sage honey. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Honey is a natural product used worldwide as an alternative to sugar, but it has wider nutritional properties because it contains a vast range of minor components of interest. Along with sugars and water, honey contains phenolic acids, flavonoids, organic acids, products of the Maillard reaction, carotenoids, amino acids and proteins, enzymes (glucose oxidase, catalase and peroxidase) and vitamins (Bogdanov, Jurendic, Sieber, & Gallmann, 2008). Lipophilic vitamins like vitamin K (Bogdanov et al., 2008) and E (Guerrini et al., 2009), and hydrophilic vitamins, mainly vitamin C(Léon-Ruiz, Vera, González-Porto, & San Andrés, 2011) and group B vitamins (Ciulu et al., 2011) have also been detected. Recently, high levels of lumichrome, the main metabolite of vitamin B 2 , were detected in thistle unifloral honey for the first time (Tuberoso, Bifulco, et al., 2011). Riboflavin plays a central role in metabolism acting as hydrogen carriers in biological redox reactions (Ball, 2006). It represents the prosthetic group of flavoproteins and the two main coenzyme forms are the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The US Recommended Dietary Allownces (RDAs) for riboflavin range from 0.4 to 1.3 mg/day for early infants and young adult males, respectively (McCormick, 2003). Riboflavin is present in milk, egg yolk, meat and liver, but it is also produced by certain bacteria in the colon. Riboflavin is stable in neutral and acid solutions, even exposed to heat and oxygen. Light exposure decomposes riboflavin to lumiflavin in alkaline solution and to lumichrome in neutral or acid solutions. Besides photochemical ac- tion, production of lumichrome from riboflavin by cleavage of the ribityl group can occur also by enzymatic mechanism. It is reported that several microorganisms present in the rhizosphere produce lumichrome that act as signal molecule for stimulating plant growth (Matiru & Dakora, 2005; Phillips et al., 1999). These results suggest that lumichrome is not just a waste metabolite, but can play an important role in the biological cycles. No biological activ- ity is reported for lumichrome in humans so far. 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.06.096 ⇑ Corresponding author. Tel.: +39 070 6758644; fax: +39 070 6758612. E-mail address: tuberoso@unica.it (C.I.G. Tuberoso). Food Chemistry 135 (2012) 1985–1990 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem