ARTICLE IN PRESS Food Chemistry Food Chemistry xxx (2005) xxx–xxx www.elsevier.com/locate/foodchem Identification of phenolic compounds in strawberries by liquid chromatography electrospray ionization mass spectroscopy Navindra P. Seeram *, Rupo Lee, H. Samuel Scheuller, David Heber Center for Human Nutrition, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA Received 15 November 2004; received in revised form 9 February 2005; accepted 9 February 2005 Abstract Strawberry (Fragaria x ananassa Duch.) fruits contain phenolic compounds that have antioxidant, anticancer, antiatherosclerotic and anti-neurodegenerative properties. Identification of food phenolics is necessary since their nature, size, solubility, degree and position of glycosylation and conjugation influence their absorption, distribution, metabolism and excretion in humans. Freeze- dried whole strawberry fruit powder and strawberry fruit extracts were analyzed by liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) methods. Phenolics were identified as ellagic acid (EA), EA-glycosides, ellagitannins, gallotan- nins, anthocyanins, flavonols, flavanols and coumaroyl glycosides. The anthocyanidins were pelargonidin and cyanidin, found pre- dominantly as their glucosides and rutinosides. The major flavonol aglycons were quercetin and kaempferol found as their glucuronides and glucosides. LC–ESI–MS/MS methods differentiated EA from quercetin conjugates since both aglycons have iden- tical molecular weights (302 g/mol). The identification of strawberry phenolics is necessary to generate standardized materials for in vitro and in vivo studies and for the authentication of strawberry-based food products. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Strawberries; Ellagic acid; Ellagitannins; Anthocyanins; Flavonols; LCMS 1. Introduction 1998). Phenolic compounds are abundant in highly col- ored berry fruits, and due to their popularity and high It is now well established that a diet high in fruits and consumption, these berries serve as one of our most vegetables is associated with a reduced risk of oxidative important dietary sources of phenolics (Kahkonen, Ho- stress mediated diseases such as cancer, cardiovascular pia, & Heinonen, 2001; Williner, Pirovani, & Guemes, and neurodegenerative diseases (Halliwell, 1994). The 2003). Berry fruits are reported to contain a wide variety health beneficial effects of fruits and vegetables are of phenolics including hydroxybenzoic and hydroxycin- attributed to their high levels of a wide variety of phyto- namic acid derivatives, anthocyanins, flavonols, flava- chemicals, of which phenolics constitute the greatest nols, condensed tannins (proanthocyanidins) and proportion. Phenolic compounds contain aromatic hydrolyzable tannins (Machiex, Fleuriet, & Billot, ring(s) bearing hydroxyl group(s) and can range from 1990). simple molecules to very large oligomers (Fig. 1). They Strawberry (Fragaria x ananassa Duch.) fruits are frequently occur naturally in glycosylated forms, which very popular among berries and are widely consumed make them more water-soluble although the higher in fresh forms and as food-products such as preserves, molecular weight oligomers are more insoluble (Bravo, jams, yogurts and ice creams. Strawberry fruits are re- ported to have antioxidant, anticancer, anti-inflamma- * Corresponding author. Tel.: +1 310 825 6150; fax: +1 310 206 5264. tory and anti-neurodegenerative biological properties E-mail address: [email protected] (N.P. Seeram). (reviewed in Hannum, 2004). Because of the reported 0308-8146/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2005.02.047 ARTICLE IN PRESS 2 N.P. Seeram et al. / Food Chemistry xxx (2005) xxx–xxx R R 2001; Chung, Lee, & Sung, 2002; Joseph et al., 1998; OH OH Stoner, Kresty, Carlton, Siglin, & Morse, 1999), and ex- HO O HO O + tract forms (Meyers, Watkins, Pritts, & Liu, 2003; See- ram, Momin, Bourquin, & Nair, 2001; Xue et al., OH OH OH O OH 2001). Due to the lack of standard methods for sample preparation, extraction and analyses, there is no general Anthocyanidins Cyanidin R = OH consensus on a standard protocol for quantitation of Flavonols Pelargonidin R = H phenolic compounds in fruits and other foods (Naczk Quercetin R = OH Kaempferol R = H & Shahidi, 2004). Quantitation of fruit phenolics is also complicated by factors such as differences in fruit culti- R1 vars, growing conditions, degree of ripeness, handling OH after harvest, etc. (Bravo, 1998). Our study is focused HO O OH on the identification and not quantitation of phenolic HO O compounds in strawberry fruits as freeze-dried whole OH OH R2 R strawberry fruit powder (SFP) and strawberry fruit Flavanols Hydroxycinnamic acid extracts (SFE). p-coumaric acid R = H (+)-Catechin (2R, 3S) R1 = OH, R2 = H Many previous methods to identify strawberry fruit phenolics were optimized for particular groups of com- OH pounds such as anthocyanins (Lopes-da-Silva, Pascual- HO Teresa, Rivas-Gonzalo, & Santos-Buelga, 2002)or O O ellagitannin-based compounds (Hakkinen, Karenlampi, HO C HO O OH O HO C O Mykkanen, Heinonen, & Torronen, 2000; Maatta-Riihi- O O O OH C HO O O OH nen, Kamal-Elin, & Torronen, 2003; Williner et al., HO O O O OH 2003). In addition, many previous methods to identify OH OH HO OH O strawberry fruit phenolics were based on spectropho- HO OH OH OH Ellagic acid tometry (Meyers et al., 2003) or high performance liquid chromatography with UV detection (HPLC–UV) [also Ellagitannin Galloyl-bis-HHDP-glucoside commonly referred to as photodiode array detection (HPLC–PDA) or diode array detection (HPLC– OH DAD)] (Gil, Holcroft, & Kader, 1997; Kosar, Kafkas, HO Paydas, & Baser, 2004; Wang and Zheng, 2001). How- O ever, HPLC–UV has its limitations since it relies on OH HO C O OH HO HO C O the comparison of characteristic absorbance wavelength O O O C O O O OH spectra, wavelength maxima (kmax), and chromato- HO C HO O O HO O graphic retention times (t ) with authentic standards. HO C O O R O O O HO OH OH C On the other hand, the use of HPLC with mass spec- O O OH HO O HO OH OH OH O trometry (HPLC–MS) detection provides useful struc- OH HO OH OH tural information and allows for tentative compound HO OH OH OH identification when standard reference compounds are unavailable and when peaks have similar t and similar Ellagitannin R Sanguiin H-6 UV-absorption spectra (Hakkinen, Karenlampi, Hei- nonen, Mykkanen, & Torronen, 1999; Lopes-da-Silva Fig. 1. Examples of phenolic compounds found in strawberry fruits. et al., 2002; Maatta-Riihinen, Kamal-Elin, & Torronen, 2004). In addition, tandem mass spectrometric (MSn) biological properties associated with strawberry fruits, techniques are useful for distinguishing compounds with the identification of their phytochemicals is necessary identical molecular weights (Mullen, Yokota, Lean, & for the evaluation of strawberry consumption on human Crozier, 2003). health. The nature, size, structure, solubility, degree and The objective of our study was to identify the pheno- position of glycosylation, and conjugation of phenolics lic compounds and generate characteristic chromato- with other compounds can influence their bioavailabil- graphic ÔfingerprintsÕ of SFP and SFE by liquid ity, absorption, distribution, metabolism and excretion chromatography electrospray ionization mass spectrom- in humans (Aherne & OÕBrien, 2002; Hollman, 2001). etry (LC–ESI–MS) methods. We also report on the use However, it is also necessary to identify strawberry of LC–MS/MS methods to differentiate between the su- phenolics in the different forms in which the fruit has gar conjugates of quercetin and ellagic acid (EA) present been studied, namely, as freeze-dried whole fruits in strawberries since both aglycons have identical molec- (Cao, Russell, Lischner, & Prior, 1998; Carlton et al., ular weights (MW 302 g/mol). Our study provides useful ARTICLE IN PRESS N.P. Seeram et al. / Food Chemistry xxx (2005) xxx–xxx 3 information required for the generation of standardized minary analyses were carried out using full scan, data strawberry materials for in vitro and in vivo studies and dependent MS/MS scanning from m/z 250–2000. The for the authentication of strawberry-based food capillary temperature was 275 °C, sheath gas and auxil- products. iary gas were 45 and 0 units/min respectively, and source voltage was 4 kV. MS/MS fragmentation was carried out with 50% energy. Zoom scan analyses were carried 2. Experimental out to determine the charge state of some of the ellagit- annin-based compounds. Identities of the compounds 2.1. Reagents were obtained by matching their molecular ions (m/z) obtained by LC–ESI–MS and LC–ESI–MS/MS with All solvents were HPLC grade and purchased from literature data (Lopes-da-Silva et al., 2002; Maatta- Fisher Scientific Co. (Tustin, CA). EA, catechin, epicat- Riihinen et al., 2004; Mullen et al., 2003). echin and quercetin standards were purchased from Sigma Aldrich Co. (St. Louis, MO). Pelargonidin and cyanidin aglycons and their respective 3-glucosides were 3. Results and discussion purchased from Chromadex Inc. (Santa Ana, CA). 3.1. General 2.2. Strawberry fruit powder (SFP) and strawberry fruit extract (SFE) HPLC–DAD–ESI–MS methods were used to analyze SFP and SFE. Common solvents used for extraction of Fresh strawberry fruits and freeze-dried whole straw- phenolic compounds from foods include water, metha- berry fruit powder (SFP) were provided by the Califor- nol, aqueous acetone, ethanol and