Analytical Methods Quantitative determination of major polyphenol constituents in pomegranate products Wenjuan Qu a , Andrew P. Breksa III b,⇑ , Zhongli Pan b,c,⇑ , Haile Ma a a School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, China b Processed Foods Research Unit, West Regional Research Center, USDA-ARS, 800 Buchanan St., Albany, CA 94710, USA c Department of Biological and Agricultural Engineering, University of California – Davis, One Shields Avenue, Davis, CA 95616, USA article info Article history: Received 24 May 2011 Received in revised form 13 October 2011 Accepted 23 November 2011 Available online 1 December 2011 Keywords: Pomegranate Gallic acid Punicalagin Ellagic acid HPLC abstract The anti-oxidant content and potential health benefits associated with consuming pomegranate and pomegranate-containing products has lead to increased consumer demand for this crop resulting in it becoming a high value crop. The potential health benefits and high anti-oxidant content of this fruit is attributed to the polyphenolic compounds it contains, including the ubiquitous phenolic acids, gallic acid and ellagic acid, and punicalagin A and punicalagin B, two polyphenolics unique to this fruit. A rapid HPLC–UV method targeting these four metabolites requiring minimal sample cleanup and offering run- times half as long as existing methods was established. Within day and inter-day run-to-run variability for the four metabolites ranged from 1.9% to 6.6% and 5.3% to 11.4%, respectively. Spike recovery percent- ages for gallic acid, punicalagin A, punicalagin B and ellagic acid were found to be 98.5%, 92.4%, 95.5%, and 96.5%, respectively. This method was applied to the evaluation of various pomegranate products, includ- ing commercial drinks, handmade juice, and marc extracts. This method may be readily used to verify the presence of pomegranate metabolites in juices, extracts, and other products. Published by Elsevier Ltd. 1. Introduction In recent years pomegranate ( Punica granatum) has become a high value crop due to increased consumer demand resulting from the potential health promoting benefits obtained through consum- ing pomegranate fruits and pomegranate containing products. Pomegranate fruits are rich in ellagitannins and much of the health promoting potential of pomegranate has been attributed to these polyphenolic compounds. Some of the main polyphenol constitu- ents found in pomegranate include punicalagins A and B, gallic and ellagic acids (Fig. 1) (Gil, Tomas-Barberan, Hess-Pierce, Holcroft, & Kader, 2000; Pérez-Vicente, Serrano, Abellán, & Gar- cía-Viguera, 2004; Seeram, Lee, Hardy, & Heber, 2005). Punicala- gins are reported to possess remarkable anti-inflammatory and anti-genotoxic properties (Chen, Li, Liu, & Lin, 2000; Kulkarni, Mahal, Kapoor, & Aradhya, 2007). Whereas antiproliferative, anti- cytotoxic, antifungal and antibacterial properties have been reported for gallic acid (Fiuza et al., 2004). Ellagic acid has been shown to possess antioxidant, anticancer and anti-atherosclerotic activities (Seeram, Lee, & Heber, 2004). In the USA, pomegranate is commercially cultivated almost exclusively in the California, USA. Of the approximate 20.5 thou- sand tons of pomegranate fruits produced annually, 75% of the har- vest is marketed as fresh fruit and the remaining 25% is processed into juice and used in making 100% juice beverages, soft drinks, confectionary products, and in the preparation of natural red food colourants (Mishkin & Saguy, 1982). Processing one ton of fruit yields approximately 322–341 L of juice and generates about 669 kg of pomegranate marc, a by-product made up of seeds and peels. In California alone, the annual production of pomegranate marc amounts to 3.4 thousand tons. Like pomegranate juice (Gil et al., 2000), pomegranate marc has also been shown to contain high levels of polyphenols (Qu et al., 2009) and thus this material is a potential source for isolating value-added antioxidants. Since the popularity of pomegranate containing fruit juices and related products with the general public stems from the presence of bioactivity of polyphenols, the ability to quantitate these com- pounds in fruits, beverages, and extracts is essential to studying their nutritional and health effects, and for proper product label- ling. From a manufacturing perspective, measuring these com- pounds in raw and finished materials is not only important because they contribute to sensorial-organoleptic attributes of products (Tiwari, O’Donnell, Patras, & Cullen, 2008), but it is becoming increasingly more important to address growing 0308-8146/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.foodchem.2011.11.106 ⇑ Corresponding authors at: Processed Foods Research Unit, West Regional Research Center, USDA-ARS, 800 Buchanan St., Albany, CA 94710, USA. Tel.: +1 510 5595898; fax: +1 510 5595849 (A.P. Breksa III), tel.: +1 510 5595861; fax: +1 510 5595851 (Z. Pan). E-mail addresses: andrew.breksa@ars.usda.gov (A.P. Breksa III), Zhongli.Pan@ars. usda.gov (Z. Pan). Food Chemistry 132 (2012) 1585–1591 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem