Research article Received: 17 April 2014

Revised: 18 June 2014

Accepted: 27 June 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3423

In-time and in-space tandem mass spectrometry to determine the metabolic profiling of flavonoids in a typical sweet cherry (Prunus avium L.) cultivar from Southern Italy Pasquale Crupi, Rosalinda Genghi and Donato Antonacci* This paper presents a comprehensive analytical methodology, based on ‘in-time’ and ‘in-space’ tandem mass spectrometry (MS) techniques, to identify and quantify flavonoid compounds in a typical Italian sweet cherry cultivar (cv. Ferrovia). Five anthocyanins, four flavan-3-ols and nine flavonols were determined by means of hyphenated high-performance liquid chromatography – multi-stage MS (HPLC-MSn) analyses (MSn up to MS4), among which quercetin-3-O-rutinoside-7-O-glucoside, kaempferol-3-O-rutinoside-7-O-glucoside, quercetin-3-O-galactosyl-rhamnoside and quercetin-3-O-coumaroylglucoside were tentatively identified in sweet cherries for the first time. Ultrafast HPLC and tandem MS (UHPLC-MS/MS) analyses through multiple reaction monitoring experiments showed that cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside were the main anthocyanins of cv. Ferrovia at maturity. Moreover, consistent levels of catechin and epicatechin as well as quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside were also found. Because flavonoids have been ascribed as potential health-promoting compounds, gathered findings provide new insight into the knowledge of the quali-quantitative profile of these phytochemicals into a widespread fruit such as sweet cherry. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: metabolic profiling; tandem mass spectrometry; flavonoids; Prunus avium L

Introduction

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* Correspondence to: Donato Antonacci, Unità di ricerca per l’uva da tavola e la vitivinicoltura in ambiente mediterraneo, CRA – Consiglio per la Ricerca e la sperimentazione in Agricoltura, Via Casamassima 148, 70010 Turi, BA, Italy. E-mail: [email protected] Dedicated to Professor Ruggero Curci (University of Bari, Italy) on the occasion of his 77th birthday. Unità di ricerca per l’uva da tavola e la vitivinicoltura in ambiente mediterraneo, CRA – Consiglio per la Ricerca e la sperimentazione in Agricoltura, Via Casamassima 148, 70010, Turi, BA, Italy

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Flavonoids (e.g. anthocyanins, flavonols and flavan-3-ols) constitute a very diverse group of secondary metabolites. Most flavonoids in plant cells are present as glycosides, in which the sugar moieties (from one to four) can be linked to hydroxyl groups, in the case of O-glycosides, or directly to carbon atoms in the ring A as happens in C-glycosides.[1] Numerous fundamental functions of this class of secondary metabolites have been ascertained; flavonoids comprise the colored pigments of many fruits[2,3] and play an important role in interactions of plants with the environment, because they can act as enzyme inhibitors, as a defense system against ultraviolet radiation exposure and insects and as chelating agents of metals that are noxious to plants.[4,5] They are also involved in photosensitization and energy transfer, morphogenesis and sex determination, photosynthesis and regulation of plant growth hormones.[6] Moreover, because there is a strong tendency for chemotaxonomically related plants to produce similar types of flavonoids, they are commonly used as chemotaxonomic markers.[7] Finally, these plant metabolites also affect the human and animal health if they are assumed with the diet. This is ascribed to their antioxidant properties[8] and their role as radical scavengers,[9,10] together with a wide spectrum of antimicrobial and pharmacological activities.[11–13] Fruits are a rich source of phenols in the diet.[14–16] In numerous areas of Europe, West Asia and South America, fruits of the Prunus species are the first fresh fruits of the season, consumed mainly non-processed and contain substantial amount of phenolic antioxidants. In particular, sweet

cherries (Prunus avium L.) are important commercially as a table fruit, and global cherry production was reported as 2 072 455 tonnes (FAO 2010).[17] It has been known since the beginning of the 20th century that sweet cherries contain substantial amounts of anthocyanins and polyphenols.[18] Total and individual contents of phenolic and anthocyanin compounds in sweet cherry cultivars have been previously reported;[3,19,20] however, the number and type of flavonols and flavan-3-ols present in this crop are less documented.[21–23] Because of the relevant activity of flavonoids and their glycosides to, and in, living organisms, being strongly dependent on their structural features, it is important to have access to rapid and reliable methods for the analysis and identification of these natural polyphenolic compounds, in all their many forms, in some widespread fruit, such as sweet cherry. In this sense, a metabolic profiling system that by definition deals with the determination of a group of metabolites either related to a specific metabolic pathway or a class of compounds is considered mandatory.[24] Determination of the absolute structure of flavonoids is rather

P. Crupi, R. Genghi and D. Antonacci difficult, and it generally implicates the use of analytical tools, such as 1H and 13C NMR-spectrometry or 1H–1H correlated spectroscopy, all requiring large amounts of purified sample.[25] Flavonoids, however, are usually present in a complex matrix of plant extracts, thus very hard to isolate in higher quantities. Coupling liquid chromatography (LC) to mass spectrometry (MS) can overcome this drawback allowing reliable identification.[26] Therefore, in this study, a comprehensive analytical method was developed, on the basis of tandem MS techniques. Flavonoid compounds in methanolic sweet cherry (cv. Ferrovia) extracts were first identified by means of hyphenated high-performance LC (HPLC), diode array detector and multi-stage MS with electrospray ionization (HPLC-DAD-ESI-MSn) analyses, with emphasis on the minor constituents present; successively, taking advantage from multiple reaction monitoring (MRM) to improve sensibility and quantification limits, an ultrafast HPLC and tandem MS (UHPLC-MS/MS) approach was also employed to quantify the main flavonoids present.

Experimental Plant material A sweet cherry cultivar (cv. Ferrovia) grown in Apulia region (Southern Italy) was used in this study. Samples were harvested at commercial maturity (first decade of June), on the basis of total soluble solids (TSS), measured as °Brix using a portable refractometer (Atago PR32, Norfolk, Virginia, USA) and titratable acidity (TA), which was determined in the juice by titration with 0.1 N of NaOH (J.T. Baker, Deventer, Holland) to a pH 7 end point and was expressed as gram of tartaric acid per liter (TSS = 17.1 °Brix; TA = 7.0 g/L), in 2011 season using 7 year-old sweet cherry trees located in Turi (longitude 40.56° E, latitude 17.12° N and altitude 280 m). The trees were trained to a central leader system and planted at a spacing of 4 m × 4 m; starting from blossoming and until the harvest, they were irrigated through a localized system (drip irrigation) with a water supply of 600 m3/ha. The trees were also fertilized, from petal falls to the end of August, with 50–60 kg/ha of nitrogen in fractionated supplies and 200 kg/ha of calcium nitrate, divided in two supplies. Then, in order to control Comstockapsis perniciosa, Archips rosanus and Tetranychus urticae, Monilia laxa and Rhagoletis cerasi, spray treatments with white mineral oil, triazoles and Phosmet (Spada 200 EC, Sariaf Gowan, Faenza, Italy), respectively, were carried out from the end of February to the veraison. A total of 5 kg of cherries were taken on the same day, from four different branches of an individual tree and mixed, and then, they were frozen in liquid nitrogen and vacuum packed in plastic bags and stored at 80 °C for further analysis. Chemicals

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Formic acid and HPLC grade water were purchased from J.T. Baker (Deventer, Holland). LC–MS grade solvent acetonitrile was purchased from Riedel-de Haën (Steinheim, Germany). Cyanidin-3O-glucoside chloride, cyanidin-3-O-rutinoside chloride, delphinidin3-O-glucoside chloride, quercetin-3-O-rutinoside, quercetin-3-Oglucoside, kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-glucoside, (+)-catechin, ( )-epicatechin, procyanidins B1 and B2 and epicatechin gallate were purchased from Extrasynthese (Genay, France). Cyanidin-3-O-sophoroside chloride and quercetin-4’-O-glucoside were purchased from Phytolab (Vestenbergsgreuth, Germany).

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Extraction of flavonoids from sweet cherry The extraction procedure for flavonoids was adapted from the method of Escarpa and González (2000).[27] Approximately, 100 g of partially defrosted sweet cherry sample were pitted, and a fruit homogenate was prepared using an IKA A11 basic homogenizer (IKA, WERKE GMBH & CO.KG, Germany). To avoid metabolite degradation, the homogenization was carried out in the dark, and the sample was kept on ice throughout the entire process (5 min). A 10-g sample of homogenate was transferred to a volumetric flask, and 10 ml of methanol solution (1% hydroxybutyl anisole – BHA), together with 50 μl of delphinidin-3-O-glucoside and epicatechin gallate, and 100 μl of isoramnethin-3-O-glucoside internal standard solutions (1000 μg/ml of methanol) were added. Then, the mixture was sonicated (BRANSON 5200) for 1 h at room temperature, and the supernatant was filtered through a custom vacuum system of filtration. The extraction procedure was repeated twice using 10 and 5 ml for 30 min, respectively, of fresh methanolic solutions. The effectiveness of repetitive extraction was tested by measuring the absorbance at 520 and 280 nm; a quantitative recovery for the analyzed compounds was obtained (Figure S1). Finally, in order to obtain a good precision under repeatability conditions, the pooled extracts were 2.5-fold concentrated under vacuum by a rotovapor Buchi-R-205 at 40 °C and stored at 25 °C until further analysis. Extractions were repeated six-folds. HPLC-DAD-MSn analysis Separation and identification of flavonoids were carried out using an HPLC 1100 (Agilent Technologies, Palo Alto, CA, USA) equipped with a model G1379A degasser, a model G1311A quaternary pump solvent delivery, a model G1316A column oven, a model G1315B DAD system and a model G2447A XCT-trap Plus mass detector (Agilent Technologies, Palo Alto, CA, USA) coupled with a pneumatic nebulizer-assisted electrospray LC–MS interface. Samples were injected onto a reversed stationary phase column, Luna C18 (150 × 2 mm i.d., particle size 3 μm, Phenomenex, USA) using a model GE1313A auto sampler (Agilent Technologies, Palo Alto, CA, USA). A pre-column, Gemini C18 5 μm (4 × 2 mm i.d., Phenomenex, USA) was fitted to protect the main column. The following gradient system was used with water/ formic acid (99 : 1, v/v) (solvent A) and acetonitrile/formic acid (99 : 1, v/v) (solvent B): 2 min, 95% A – 5% B; 10 min, 87% A – 13% B; 25 min, 85% A – 15% B; 30 min, 78% A – 22% B; 50 min 78% A – 22% B; 55 min 5% A – 95% B; 65 min 5 % A – 95% B; 66 min 95% A – 5% B; stop time 80 min. The column was kept at 40 °C, the flow was maintained at 0.2 ml/min and the sample injection was 3 μl. The flow rate and the elution program were controlled by an LC ChemStation 3D software (Hewlett-Packard, USA). Wavelenghts for the ultraviolet-visible (UV-vis) detection were set at 520, 360 and 280 nm, and spectrophotometric spectra of flavonoids were registered from 250 to 650 nm. Both positive and negative ESI were used for ionization of molecules with capillary voltage at 4000 V and skimmer voltage at 40 V. The nebulizer pressure was 30 psi, and the nitrogen flow rate was 8 l/min. Temperature of drying gas was 350 °C. In the full-scan mode, the monitored mass range was from mass to charge ratio (m/z) 100 to 1200 at a scan speed of 13000 Da/s. MSn was performed by using helium as the collision gas at a pressure of 4.6 × 10 6 mbar. Collision-induced dissociation spectra were obtained with an isolation width of 4.0 m/z for precursor

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Flavonoid pattern in sweet cherry cv. ‘Ferrovia’ ions and a fragmentation amplitude of 0.8 V (MSn up to MS4). Compound identification was achieved by combining different information: positions of absorption maxima (λmax), the elution order and mass spectra were compared with those from pure standards and interpreted with the help of structural models already hypothesized in the literature.[20,21,28] UHPLC-ESI-MS/MS analysis The ultrafast chromatographic system consisted of a capillary HPLC 1290 Infinity (Agilent Technologies, Palo Alto, CA, USA) equipped with a model G4220A binary pump solvent delivery, a model G1316A thermostatic column compartment, a model G4226A auto sampler and a model G6430A triple quadrupole QQQ mass detector (Agilent Technologies Palo Alto, CA, USA) coupled with a pneumatic nebulizer-assisted electrospray LC– MS interface. A Zorbax column SC-C18 (50 × 2.1 mm i.d., particle size 1.8 μm, Agilent Technologies) was used, with the following gradient system: water/formic acid (99 : 1, v/v) (solvent A) and acetonitrile/formic acid (99 : 1, v/v) (solvent B), 0.8 min, 95% A – 5% B; 2.1 min, 90% A – 10% B; 5.6 min, 88% A – 12% B; 8 min, 81% A – 19% B; 9.2 min 81% A – 12% B; 11.2 min 5% A – 95% B; 12.8 min 5% A – 95%; 13.2 min 95% A – 5%; stop time 15 min. The column was kept at 60 °C, the flow was maintained at 0.5 ml/min and the sample injection was 1.1 μl. Both positive and negative ESI modes were used for ionization of molecules with capillary voltage at 4000 V. Nitrogen was used both as drying gas at a flow rate of 8 l/min and as nebulizing gas at a pressure of 30 psi. Temperature of drying gas was 350 °C. In the full-scan (MS) and product ion (MS/MS) modes, the monitored mass range was from m/z 100 to 1200. Typically, two runs were performed during the UHPLC-ESI-MS analysis of each sample. First, an MS full-scan acquisition was performed to obtain preliminary information on the predominant m/z ratios observed during the elution. An MS/MS full-scan acquisition was then performed: quadrupole 1 filtered the calculated m/z of each compound of interest, while quadrupole 3 scanned for ions produced by nitrogen collision of these ionized compounds in the chosen range at a scan time of 500 ms/cycle. All data were acquired and processed using MASSHUNTER software (version B.01.04; Agilent Technologies). The optimized parameters (fragmentor voltage and collision energy) for each compound together with the mass transitions adopted for MRM are listed in Table S1. To evaluate linearity, calibration curves with five/seven concentration points for each compound were prepared separately. Calibration was performed by linear regression of peak-area ratios of the flavonoids to the relative internal standard versus the respective standard concentration. The precision of the method was determined by calculating the intraday and interday repeatability, expressed as standard deviation (SD) (σ) in terms of retention times and peak width (W1/2), by injection of six different replicates of extracts in the same day or during three consecutive days. The detection limit (LOD) and quantification limit (LOQ) were calculated on the basis of chromatograms and defined as signal-to-noise (six times SD of baseline) ratio of 3 and 10, respectively (Table 1). Statistical analysis

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Extraction of polyphenols depends on their diffusion into the extraction solvent, which is determined by either their structure or their interactions with other fruit components. However, owing to the high selectivity of MS, especially in combination with LC and MS/MS, no extensive sample preparation is required; the unique limitation is that the extraction procedures must allow quantitative recovery of flavonoids, while avoiding any chemical modification and degradation.[5] Thus, literature gathers various extraction conditions (time, solvent) according to the accessibility of polyphenols in the different matrices; in particular, lower molecular weight polyphenols, such as flavonoids, are well extracted with methanol.[29] Therefore, in agreement with previous studies,[21,27] a non-acidified methanolic medium together with optimum extraction times and solid-to-liquid ratios were chosen in this work to provide an extraction of the polyphenol pool as complete as possible while limiting their degradation and avoiding the occurrence of artifact compounds. On the basis of the type of searched compounds, ESI ionization analyses both in positive (in the case of anthocyanins) and negative (in the case of flavan-3-ols and flavonols) ion mode were carried out; moreover, the combined use of both ionization modes, by focusing on the formation of alkali metal adducts, i.e. [M + Na]+, often identified in the first-order mass spectra, was chosen to give extra certainty to the molecular mass determination, especially for the minor compounds where the noise level is much higher. Representative MSn spectra of compounds of interest were also thoroughly discussed in respect to the presence/absence or the abundance level of distinct fragment ions, which can help to determine important structural features. Characterization of flavan-3-ols and flavonols in the sweet cherry extract Figure 1(a) shows the extracted ion chromatograms of a sweet cherry (cv. Ferrovia) methanolic extract recorded in negative-ion mode by the ion trap spectrometer device. The gradient profile used in this work allowed the separation of the compounds with a retention that, in general, followed the expected reversedphase pattern of flavonols O-glycosides > favan-3-ols and proanthocyanidins. To investigate the fragmentation pathways of these compounds, the negative-ion mode was chosen because it appeared more sensitive for ESI-MS analysis of flavonols and flavan-3-ols.[30] The mass spectra of catechin (peak 2) and epicatechin (peak 7) showed the deprotonated molecule [M H] (m/z 289) and the product ion at m/z 245 generated from the loss of a –CH2CHOH group, as previously described.[31] Peaks 1 and 5 corresponded to dimeric B-type procyanidins, because they were characterized by the same [M H] (m/z 577) and the fragment ions at m/z 451, 425 and 407 derived from heterocyclic ring fission, retroDiels–Alder fission and consecutive water loss, respectively. Moreover, the typical m/z 289 ion (the deprotonated catechin or epicatechin unit) generated from interflavinic bond cleavage was also revealed in their MS2 spectra.[32] Therefore, from matching with reference standards, they were identified as procyanidin B1 and procyanidin B2, respectively (Table 2). Five quercetin derivatives were identified on the basis of the typical fragmentation pattern of the aglycone form; indeed, as showed in the MS3 spectra of the peaks 12, 13, 14 and 17 as well as in the MS4 spectrum of the peak 10, the fragmentation of the

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Means and SDs of the raw data and regression analysis of calibration samples were carried out using STATISTICA 6.0 software package (StatSoft Inc., Tulsa, OK, USA).

Results and discussion

2

MRM, multiple reaction monitoring; R , determination coefficient; LOD, limit of detection as signal/noise = 3; LOQ, limit of quantification as signal/noise = 10; RT, retention time; W1/2, peak widths.

0.141 ± 0.009 0.151 ± 0.008 0.185 ± 0.012 0.169 ± 0.011 0.27 ± 0.02 4.504 ± 0.009 4.542 ± 0.008 6.16 ± 0.11 6.179 ± 0.011 6.29 ± 0.12 0.147 ± 0.002 0.154 ± 0.003 0.190 ± 0.006 0.176 ± 0.004 0.252 ± 0.004 4.567 ± 0.007 4.608 ± 0.007 6.255 ± 0.019 6.269 ± 0.017 6.40 ± 0.02 172.5 23.6 14.9 8.8 11.2 51.7 7.1 4.5 2.6 3.4 0.9978 0.9992 0,9989 0,9993 0,9991 0.010 ± 0.005 0.005 ± 0.003 0.090 ± 0.011 0.012 ± 0.008 -0.005 ± 0.011 0.625–10 0.625–10 0.625–10 0.625–10 0.625–10

0.081 ± 0.001 0.0773 ± 0.0006 0.226 ± 0.002 0.212 ± 0.002 0.253 ± 0.002

0.107 ± 0.005 0.091 ± 0.004 0.096 ± 0.005 0.096 ± 0.003 1.06 ± 0.03 1.26 ± 0.04 1.93 ± 0.04 2.20 ± 0.04 0.105 ± 0.007 0.092 ± 0.003 0.099 ± 0.005 0.098 ± 0.004 1.102 ± 0.016 1.302 ± 0.010 1.987 ± 0.016 2.253 ± 0.012 230.0 54.4 40.1 20.4 69.0 16.3 12.0 6.1 0.9983 0.9988 0.9990 0.9987 0.1046 ± 0.0012 0.017 ± 0.03 0.035 ± 0.005 0.014 ± 0.003 1–10 1–10 1–10 1–10

0.046 ± 0.007 0.0431 ± 0.0004 0.0940 ± 0.0008 0.0441 ± 0.0004

0.112 ± 0.005 0.112 ± 0.002 0.111 ± 0.001 2.16 ± 0.04 2.37 ± 0.04 2.64 ± 0.03 0.115 ± 0.007 0.113 ± 0.003 0.112 ± 0.002 2.198 ± 0.015 2.419 ± 0.011 2.680 ± 0.009 9.7 17.9 55.3 2.9 5.4 16.6 0.9989 0.9964 0.9955 0.058 ± 0.012 -0,07 ± 0,06 0.07 ± 0.06 0.299 ± 0.002 0.7619 ± 0.011 0.0010 ± 0.0002 0.1–10 0.1–10 100–500

Anthocyanins Cyanidin-3-O-sophoroside Cyanidin-3-O-glucoside Cyanidin-3-O-rutinoside Flavan-3-ols Procyanidin B1 (+)-Catechin Procyanidin B2 ( )-Epicatechin Flavonols Quercetin-3-O-rutinoside Quercetin-3-O-glucoside Kaempferol-3-O-glucoside Kaempferol-3-O-rutinoside Quercetin-4′-O-glucoside

W1/2(min) ± σ RT(min) ± σ W1/2(min) ± σ

Interday assay Intraday assay

RT(min) ± σ LOQ (ng/ml) LOD (ng/ml) Concentration range (μg/ml)

Slope ± σ

Intercept ± σ

R

2

Compound

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Table 1. Validation parameters, linearity, repeatability (r), limit of detection and limit of quantification for multiple reaction monitoring ultrafast high-performance liquid chromatography and tandem mass spectrometry analyses

P. Crupi, R. Genghi and D. Antonacci

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ion at m/z 301 gave rise to the most diagnostic fragments for quercetin identification represented by the cleavage of two C–C bonds (namely 1 and 2 bonds) of the C-ring, i.e. the ions 1,2 B0 at m/z 121 and 1,2A0 at m/z 179, followed by the further consecutive loss of CO (m/z 151) and CO2 (m/z 107) moieties (Figure S2).[30] Because of the presence of the same [M + H]+ and [M + Na]+ ions in their MS1 spectra together with the diagnostic neutral losses of 162 and 120 atomic mass unit (u), ascribable to a hexose moiety,[5] in the MS2 spectrum of the deprotonated molecule [M H] at m/z 463 (Table 2), peaks 14 and 17 were recognized as quercetin glucosides; in particular, because deprotonated flavonol glycosides (prevalently at C3 position) can undergo both a collision-induced homolytic and heterolytic cleavage of the O-glycosidic bond producing deprotonated radical aglycone, (Y0 H) ·, and aglycone, Y0 , product ions,[33] the two compounds corresponded to quercetin-3-O-glucoside and quercetin-4′-O-glucoside (spiraeoside), respectively, as further confirmed from matching their retention times and spectral behavior with reference standards (Figure S3). Peaks 12 and 13 were identified as quercetin diglycosides having the same ions [M H] and [M + H]+ together with the common neutral loss of 308 u (m/z 609 → m/z 301); furthermore, as no intermediate quercetin-hexose or quercetin-rhamnose MS2 ions were detected, it was concluded that the cleaved sugar is a rutinose-type fragment (i.e. glucose-rhamnose, which is known not to fragment into its constitutive sugars). In addition, because of the very low abundance of the ion Y1 at m/z 463 and the absence of Z1 at m/z 445, a 1 → 2 sugar linkage type could be excluded.[26] Therefore, according to their chromatographic behavior and UV-vis spectra (Table 2), compound 13 was quercetin-3-O-rutinoside, as also confirmed by reference with authentic standard, while the earlier eluting compound 12 could be named as quercetin-3-O-galactosylrhamnoside, in agreement with previous literature data.[34] Finally, peak 10 was ascribed to a quercetin 3,7 triglycoside derivative because the MS2 spectrum of its deprotonated molecule ([M H]- = m/z 771) presented three fragments at m/z 609 (Y70 ), 463 (Y30 ) and 301 (Y0 ) maybe derived from the loss of a hexose, rutinose and hexose-rutinose group, respectively. Showing a very similar fragmentation pattern to that of the MS2 spectrum of quercetin-3-O-rutinoside (peak 13, Table 2), the MS3 product ion spectrum of the fragment at m/z 609 provided sufficient structural information to indicate the linkage of the rutinoside moiety to C3′ of the B-ring; furthermore, the relative abundance of Y70 ion was higher than that of Y30 ion, suggesting that the neutral loss of the 7-O-glycosyl residue was more favorable than that of the 3-O-glycosyl residue (Fig. 2) and was consistent with the hexose residue located at the 7-O position and the rutinose residue located at the 3-O position.[35] Furthermore, unambiguous information about the glucoside position was also obtained from the MS2 fragmentation spectrum of the sodiated molecule [M + Na]+[36]; indeed, the absence of a C-ring fragment containing the sugar residue clearly excluded 4′O-glucosylation (data not shown). Therefore, this compound was tentatively identified as quercetin-3-O-rutinoside-7-O-glucoside. It is worth noting that also peak 18 showed an [M H]- ion at m/z 609, typical of quercetin diglycoside derivatives; however, from its fragmentation pattern as well as chromatographic behavior in line with previous statements,[5,37] this compound was tentatively identified as quercetin-3-O-coumaroyl-glucoside because of the MS2 fragments at m/z 463 (base peak – loss of coumaroyl group) and 301 (loss of coumaroylhexose). The exact location of the acyl group on the glycosidic part is difficult to

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Flavonoid pattern in sweet cherry cv. ‘Ferrovia’ spectrum of kaempferol-3-O-rutinoside and the relative abundance of Y70 ion was higher than that of Y30 ion, suggesting that the glucose residue was located at the 7-O position, and the rutinose residue was located at the 3-O position (Fig. 3).[35]

Characterization of anthocyanins in the sweet cherry extract As showed in Table 2 and Fig. 1(c), four anthocyanidins diglycosides (peaks 3, 6, 8 and 9) can be distinguished by referring to their MS spectra; in particular, they were identified as 3O-diglycoside derivatives because the molecular ion [M]+ yielded an MS2 ion corresponding to the aglycone, as base peak, and the very low fragment due to the loss of a monoglycosyl moiety. In contrast, this fragment should have been higher in the case of diglycosides with the glycosyl groups at two loci rather than one, such as anthocyanidins-3,5-O-diglycosides.[3] Namely, compounds 3 and 6 were identified as cyanidin-3-Osophoroside and cyanidin-3-O-rutinoside, while compounds 8 and 9 were attributed to pelargonidin-3-O-rutinoside and peonidin-3O-rutinoside as corroborated from matching their spectral and chromatographic characteristics with reference standards and/or literature statements.[3,40] Finally, peak 4 was assigned to cyanidin3-O-glucoside on the basis of the diagnostic sodiated [M + Na]+ and molecular ion [M]+ at m/z 471 and 449, respectively, together with the typical MS2 fragment at m/z 287 corresponding to cyanidin aglycone. The identity of the compound was also confirmed through a synthetic standard.

Flavonoids content in the sweet cherry cv. Ferrovia

Figure 1. Extracted ion chromatogram of (a) flavan-3-ols, (b) flavonols and (c) anthocyanins recorded in negative and positive ionization modes, n respectively, by HPLC-MS ion trap spectrometric device.

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define on the basis of MS data, but they appear to be predominantly located at the six position of the hexose moiety although other positions cannot be excluded.[38] Peaks 11, 15 and 16 were recognized as kaempferol derivatives thanks to the appearance in the MS2 spectra of their deprotonated molecules of the diagnostic radical aglycone, (Y0 H) · and aglycone, Y0 product ions at m/z 285 and 284, respectively, together with the fragments at m/z 257, 229 and 213 due to the neutral loss of CO and CO2 molecules from kaempferol aglycone (Figure S4).[33,39] By comparing their chromatographic and spectral characteristics with reference standards, compounds 15 and 16 were named kaempferol-3-O-rutinoside and kaempferol-3-Oglucoside, respectively; while compound 11 was tentatively identified as kaempferol-3-O-rutinoside-7-O-glucoside because the MS3 product ion spectrum of the fragment at m/z 593 showed a very similar fragmentation pattern to that of the MS2

The individual acquisition parameters of the triple quadrupole tandem mass spectrometer for the commercially available target compounds as well as the internal standards, chosen for each flavonoid family, are summarized in Table S1. Q1 is the precursor ion mass and Q3 the fragment (product) ion mass of interest; the fragmentor voltage, collision energy and ionization sign were optimized for each analyte. Even with detection by coupled tandem MS, there are still metabolites with identical or indistinguishable molecular and fragment masses because of their structural resemblance; of course, chromatographic separation is still fundamental in these cases (Fig. 4). Therefore, catechin and epicatechin had the same precursor and product ions (289 and 245 m/z) but could be distinguished chromatographically (e, g), and the discrimination of procyanidin isomers B1 and B2 was possible by their different retention times (d, f); moreover, quercetin-3-O-glucoside and quercetin-4′-O-glucoside, with indistinguishable ion masses (463 m/z) and fragments (301 m/z), were separated by the HPLC run (i, n). In line with the choice of the main figures of merit reported elsewhere,[21] the optimized UHPLC-MS/MS method was validated for the target analysis of 12 flavonoids in terms of linearity, LOD, LOQ and precision (Table 1). The calibration curves were obtained by the internal standard method to give determination coefficients (R2), which were generally next to 0.999; moreover, except for procyanidin B1 and quercetin-3-O-rutinoside, the detection and quantification limits were found under 20 and 60 ng/ml, respectively. As regards precision tests, intra-assay and inter-assay coefficient of variation were calculated, with retention times averaged 0.52% and 1.6%, respectively, while corresponding measures for W1/2 were 3.3% and 5.4%, respectively; these results indicate that it was possible to generate accurate

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Quercetin-3-O-coumaroylglucoside

18

45.5

37.9

36.7 37.7

33.0

30.9

29.9

23.5

14.3 16.3 16.8 18.1 18.4 18.6 19.9 19.9 21.1 23.1

+

282, 332

350 282, 330

354

354

355

330, 266, 282sh

279 282 516 516 279 516 282 520 520 344

λmax (nm)

+

633.1

487.1

617.1 471.1

487.1

633.1

633.1

779.2

601.1 313.1 633.1 471.1 601.1 617.1 313.1 601.1 631.1 795.1

[M + Na] (m/z)

+

611.0

465.0

595.1 449.0

465.0

611.1

611.0

757.1

579.1 291.1 611.1 449.1 579.1 595.1 291.1 579.1 609.1 773.1

+

[M] or [M + [M + H] (m/z)

609.2

462.9

593.0 446.9

462.9

609.0

609.0

755.1

577.0 288.9 609.0 447.0 577.0 593.1 288.9 577.1 607.1 771.1

H] (m/z) 2

+

H] ,

MS [577.0]: 559.3 (13.0), 451.1 (24.2), 425.1 (100), 407.0 (69.9), 289.0 (22.7) 2 MS [288.9]: 270.9 (4.3), 244.9 (100), 204.9 (21.9), 178.9 (13.7) 2 MS [611.1]: 449.2 (2.2), 287.1 (100) 2 MS [449.1]: 287.2 (100), 236.2 (2.5) 2 MS [577.0]: 559.3 (4.3), 451.0 (15.9), 425.0 (100), 407.1 (65.3), 289.0 (18.8) 2 MS [595.1]: 449.3 (21.1), 287.1 (100), 228.0 (3.8) 2 MS [288.9]: 270.9 (4.1), 244.9 (100), 204.9 (19.4), 178.9 (16.2) 2 MS [579.1]: 433.3 (8.7), 271.1 (100) 2 MS [609.1]: 463.2 (9.2), 301.2 (100), 286.2 (30.6) 2 MS [771.1]: 609.2 (100), 463.1(4.7), 300.9(4.7), 299.9(0.2) 3 MS [771.1 → 609.2]: 591.2(1.4), 463.2(0.4), 343.0(4.5), 325.1(1.0), 301.0(100), 300.0 (32.4), 254.9(2.8) 4 MS [771.1 → 609.2 → 301.0]: 273.0(14.2), 271.0(36.3), 257.0(8.1), 255.1(34.5), 229.0(5.9), 210.9(1.8), 193.1(3.7), 179.1(100), 151.1(57.3), 121.2(1.9), 107.3(2.1) 3 MS [463.1]: 301.0 (100) 2 MS [755.1]: 593.2 (100), 447.0 (14.3), 284.9 (2.9) 3 MS [755.1 → 593.2]: 327.0(2.8), 284.9 (100), 284.0 (5.5), 257.0(6.5), 229.0(3.3), 212.9(1.5) 3 MS [755.1 → 447.0]: 326.8 (3.2), 284.9 (100), 283.8 (10.0) 2 MS [609.0]: 590.9(5.6), 540.9(4.9), 463.3(0.2), 343.0 (2.6), 325.3(0.2), 301.1(100), 300.0(15.0), 257.1(19.3) 3 MS [609.0 → 301.1]: 273.0(2.4), 270.9(1.0), 257.1(100), 255.0(0.5), 229.0(0.4), 179.1(3.2), 151.1(0.8), 121.2(0.5), 107.1(0.5) 2 MS [609.0]: 591.1(1.1), 463.2(0.4), 343.1 (6.0), 325.1(0.2), 301.1(100), 300.0 (31.4), 255.1(5.0) 3 MS [609.0 → 301.1]: 273.1(14.5), 271.0(54.1), 257.1(12.9), 255.1(36.5), 229.1(5.4), 211.1(1.4), 193.1(4.6), 179.0(100), 151.1(56.9), 121.2(1.7), 107.3(3.6) 2 MS [462.9]: 444.7 (1.3), 342.9 (1.2), 300.9 (100), 299.9 (24.5), 178.8 (3.1), 150.9 (1.5) 3 MS [462.9 → 300.9]: 272.9(19.5), 270.9(79.5), 256.9(12.8), 254.9(34.5), 228.9(11.1), 210.9(6.0), 192.9(10.6), 178.9(100), 150.9(62.9), 121.2(4.5), 107.0(4.2) 2 MS [593.0]: 326.9(2.2), 284.9 (100), 283.9 (5.3), 256.9(2.7), 228.9(2.0), 213.0(1.0) 2 MS [446.9]: 326.9(15.9), 294.8(1.0), 284.9 (77.5), 283.9 (100), 254,9(27.2), 226.9(3.4), 150.8(3.7) 3 MS [446.9 → 284.9]: 255.0(100), 227.0(14.4), 212.9 (2.1) 2 MS [462.9]: 444.7 (3.5), 300.9 (100), 299.9 (2.0), 178.9 (1.1) 3 MS [462.9 → 300.9]: 256.8(19.5), 229.0(6.7), 193.0(5.7), 178.9(71.6), 150.9(100) 2 MS [609.2]: 590.7(5.9), 562.7(5.3), 540.8(5.3), 463.1(100), 447.1(1.3), 301.0(21.8) 3 MS [609.2 → 463.1]: 300.9(100)

n

MS experiments m/z (% base peak)

RT, retention time; λmax, absorption maximum wavelength; [M + Na] , sodiated molecule; [M] , molecular ion, only present in the anthocyanidins MS spectra; [M + H] , protonated molecule; [M n th deprotonated molecule; MS , n -generation product ions.

Quercetin-4′-O-glucoside (Spiraeoside)

Quercetin-3-O-glucoside

14

17

Quercetin-3-O-rutinoside

13

Kaempferol-3-O-rutinoside Kaempferol-3-O-glucoside

Quercetin-3-O-galactosyl-rhamnoside

12

15 16

Kaempferol-3-O-rutinoside-7-O-glucoside

11

Procyanidin B1 Catechin Cyanidin-3-O-sophoroside Cyanidin-3-O-glucoside Procyanidin B2 Cyanidin-3-O-rutinoside Epicatechin Pelargonidin-3-O-rutinoside Peonidin-3-O-rutinoside Quercetin-3-O-rutinoside-7-O-glucoside

1 2 3 4 5 6 7 8 9 10

RT (min)

) characteristics of anthocyanins, flavonols and flavan-3-ols in mature sweet cherry (cv. Ferrovia)

Compound

+/

Peak

Table 2. HPLC-DAD-MS (ESI

P. Crupi, R. Genghi and D. Antonacci

J. Mass Spectrom. 2014, 49, 1025–1034

Flavonoid pattern in sweet cherry cv. ‘Ferrovia’

2

3

2

3

Figure 2. Negative-ion ESI–MS and MS spectra obtained for quercetin-3-O-rutinoside-7-O-glucoside.

Figure 3. Negative-ion ESI–MS and MS spectra obtained for kaempferol-3-O-rutinoside-7-O-glucoside.

J. Mass Spectrom. 2014, 49, 1025–1034

summarized in Table 3. Similarly to other varieties, cyanidin-3O-rutinoside and cyanidin-3-O-glucoside were the major anthocyanins found at maturity; in particular, their ratio (~1300) was higher than that determined in previous researches on Spanish

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1031

and reproducible data with the described multi-level method for flavonoids analysis. The flavonoids content, expressed as mg/100 g of fresh weight (fw), in the studied sweet cherry cultivar ‘Ferrovia’, are

P. Crupi, R. Genghi and D. Antonacci

Figure 4. Multiple reaction monitoring chromatograms of flavonoids recorded in negative and positive ionization modes through UHPLC-MS/MS triple quadrupole device. Table 3. Flavonoid content of the sweet cherry (Prunus avium L.) ‘cv. Ferrovia’ Compound Anthocyanins Cyanidin-3-O-sophoroside Cyanidin-3-O-rutinoside Cyanidin-3-O-glucoside Flavan-3-ols Procyanidin B1 Catechin Procyanidin B2 Epicatechin Flavonols Quercetin-3-O-rutinoside Quercetin-3-O-glucoside Kaempferol-3-O-glucoside Kaempferol-3-O-rutinoside Quercetin-4′-O-glucoside a

ma ± σb

0.023 ± 0.004 642 ± 16 0.50 ± 0.05 0.121 ± 0.006 0.98 ± 0.07 0.156 ± 0.009 0.83 ± 0.05 1.78 ± 0.05 0.078 ± 0.002 0.0349 ± 0.0005 0.502 ± 0.006 0.006 ± 0.002

1032

Means of six replicates expressed as mg/100 g of fresh weight (fw). Standard deviation.

b

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and Slovenian sweet cherries (ranging between 10 and 50), to demonstrate the preferential accumulation of the cyanidin diglycoside in ‘cv. Ferrovia’.[21,41] Moreover, a significant level (0.023 ± 0.004 mg/100 g fw) of cyanidin-3-O-sophoroside was quantified. Consistent level of epicatechin, comparable with French and Iranian cherries,[20] together with catechin, procyanidin B1 and procyanidin B2, were also revealed. With regard to flavonols, the main compound present was quercetin-3-O-rutinoside (rutin), at concentration very close to that found in Croatian varieties,[42] and kaempferol-3-O-rutinoside, followed by lower quantities of kaempferol-3-O-glucoside and quercetin-3-O-glucoside, while quercetin-4′-O-glucoside was found in trace.

Conclusions An optimized metabolic profiling approach allowed, in this work, the efficient determination of flavonoids from the sweet cherry ‘cv. Ferrovia’. HPLC separations coupled with MS tandem techniques made possible the identification of five anthocyanins (cyanidin, pelargonidin and peonidin diglycosides), four flavan-

Copyright © 2014 John Wiley & Sons, Ltd.

J. Mass Spectrom. 2014, 49, 1025–1034

Flavonoid pattern in sweet cherry cv. ‘Ferrovia’ 3-ols (procyanidins and catechins) and nine flavonols (quercetins and kampferols derivatives), mainly based on their sodiated, protonated and deprotonated molecules and collision-induced dissociation-MSn experiments, as well as the quantification of the main compounds identified by means of MRM analyses performed through a triple quadrupole spectrometer. Because flavonoids have been ascribed as potential health-promoting compounds, gathered findings provide new insight into the knowledge of the quali-quantitative profile of these phytochemicals into a widespread fruit such as sweet cherry. Acknowledgements This study was supported by grant from Apulia Region (PO FESRFSE 2007-2013-Project TEGUVA cod.61/09), the Italian Ministry of University and Research-MIUR (PON ‘R&C’ 2007–2013 Project ONEV – cod.00134/2011).

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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.

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