Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring

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Abstract

A systematic method for anthocyanin identification using tandems mass spectrometry (MS/MS) coupled to high-performance liquid chromatography (HPLC) with photo-diode array detection (PDA) was developed. Scan for the precursor ions of commonly found anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin, and peonidin) using LC/MS/MS on a triple quadrupole instrument allows for the specific determination of each category of anthocyanins. Further characterization of each anthocyanin was performed using MS/MS product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring (SRM). The method was demonstrated for analysis of anthocyanins in black raspberries, red raspberries, highbush blueberries, and grapes (Vitis vinifera). Previous reported anthocyanins in black raspberries and red raspberries are confirmed and characterized. Common-neutral-loss analysis allows for the distinction of anthocyanin glucosides or galactoside and arabinosides in highbush blueberries. Separation and identification of anthocyanin glucosides and galactosides were achieved by LC/MS/MS using SRM. Anthocyanin isomers such as cyanidin sophoroside and 3,5-diglucoside were differentiated by their fragmentation pattern during product-ion analysis. Fifteen anthocyanins (all possible combinations of five anthocyanidins and three sugars) were characterized in highbush blueberries. Pelargonidin 3-glucoside and pelargonidin 3,5-diglucoside were detected and characterized for the first time in grapes. The present approach allows mass spectrometry to be used as a highly selective detector for rapid identification and characterization of anthocyanins and can be used as a sensitive procedure for screening anthocyanins in fruits and vegetables.

Introduction

Anthocyanins are a group of O-glycosides of 3,5,7,3′-tetrahydroxyflavylium cation responsible for the red, blue, and violet colors of most berries and other fruits and vegetables [1]. There is an increasing interest in anthocyanins because of their use as natural food colorants [2], [3] and potential health-promoting properties. Numerous studies have shown the positive therapeutic effects of anthocyanins such as antioxidative [4], anti-inflammatory [5], DNA cleavage and cardiovascular protective properties [6]. In addition, anthocyanin composition of many fruits is distinctive and anthocyanin profiles have been used as fingerprint to clarify plants and to detect the adulteration of fruit juices [7]. Cyanidin (3,5,7,3′,4′-pentahydroxyflavylium), delphinidin (3,5,7,3′,4′,5′-hexahydroxyflavylium), malvidin (3,5,7,4′-tetrahydroxy-3′,5′-dimethoxyflavylium), pelargonidin (3,5,7,4′-tetrahydroxyflavium), peonidin (3,5,7,4′-tetrahydroxy-3′-methoxyflavylium), and petunidin (3,5,7,3′,4′-pentahydroxy-5′-methoxyflavylium) are the six most commonly found anthocyanin aglycones (Fig. 1). However, different types and numbers of sugars that are conjugated to the aglycones form numerous structures of anthocyanins and as such more than 600 different anthocyanins have been isolated from plants to date [8]. The most prevalent glycosylation in anthocyanins is glucose, however, rhamnose, galactose, xylose, and arabinose are also present in anthocyanins [2]. In addition, many anthocyanins have sugar residues acylated with aromatic or aliphatic acids such as p-coumaric, caffeic, ferulic substituents, etc. (Fig. 1).

Numerous methods have been developed for anthocyanin characterization. The most commonly used techniques are high-performance liquid chromatography (HPLC) coupled to photodiode-array detection [7], liquid chromatography–mass spectrometry (LC/MS) using continuous-flow fast atom bombardment (CF-FAB) [9], [10], electrospray ionization (ESI) [11], [12], [13], [14], atmospheric pressure chemical ionization (APCI) [15], and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) techniques [16]. Tandem mass spectrometry (MS/MS), particularly product-ion analysis that acquires a mass spectrum of the product ions produced from the fragmentation of the selected precursor ion, has been extensively used for identification and characterization of anthocyanins [11], [17]. However, other tandem mass spectrometric techniques such as precursor-ion analysis and common-neutral-loss analysis are particularly important for analysis of mixtures and screening for the presence of specific compounds in complex matrices [18]. A precursor-ion analysis detects all the precursor ions in a sample that fragment to a common product ion, whereas a common-neutral-loss analysis detects those precursor ions that fragment to product ions with a common difference in m/z produced by loss of a neutral fragment. Quadrupole mass spectrometers have been estimated to be the most widely used devices among mass analyzers in chemical research and industrial laboratories [19]. The implementation of tandem mass spectrometric techniques using a quadrupole mass analyzer facilitates the conduction of various MS/MS experiments. Furthermore, the precursor-ion analysis on a triple quadrupole mass spectrometer can specifically detect the precursors of a given indicative fragment and can be used as filters for chemical noise, significantly increasing the sensitivity of detection [20].

In this study, we report the use of precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring (SRM) tandem mass spectrometry to screen known anthocyanins in biological samples. These techniques are demonstrated in the identification and characterization of anthocyanins in fruit samples of black raspberries (Rubus occidentalis), red raspberries (Rubus idaeus), highbush blueberries (Vaccinium corymbosum), and grapes (Vitis vinifera) since these fruits contain a complete mixture of six types of anthocyanins.

Section snippets

Materials and chemicals

Black raspberries (R. occidentalis), red raspberries (R. idaeus), and blueberries (V. corymbosum) used for a clinical study were purchased from the Dale Stokes Berry Farm (Wilmington, OH). Fruit samples were ground using a Brown pulper-finisher (Brown International Corp., Covina, CA) and frozen at −20 °C before being lyophilized using a Virtis freeze-drying unit (Virtis Company, Gardiner, NY). A commercial Rubired grape extract (V. vinifera) was provided by Polyphenolics (Madera, CA).

Results and discussion

The methodology was established based on direct LC/MS/MS analysis of a mixture of four standard cyanidin anthocyanins (∼3 μmol/mL each). Screening the precursors of m/z 287 (cyanidin) detected four ions at m/z 449, 581, 595, and 611 which corresponded to the molecular cations of cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3-rutinoside, and cyanidin 3,5-diglucoside, respectively. Common-neutral-loss analysis by monitoring loss of glucose (162 U), rhamnose (146 U), and xylose (132 U) as

Conclusion

The data presented here demonstrate that the combinational use of precursor-ion analysis, common-neutral-loss analysis, product-ion analysis, and SRM on a Q1qQ2 instrument is a viable technique for screening anthocyanins in complex matrices. The use of HPLC coupled to ESI/MS/MS allowed us to identify and characterize a number of anthocyanins in fruit samples. By employing these techniques, we have confirmed and characterized previously reported anthocyanins in black raspberries and red

Acknowledgment

This study was supported by a special research grant for dietary intervention from the U.S. Department of Agriculture.

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