Flavonoids detection by HPLC-DAD-MS-MS in lemon juices from Sicilian cultivars

High-performance liquid chromatography-diode array detector (HPLC-DAD) coupled with electronspray mass spectrometry (ESI-MS) was used to detect the flavonoid profile in lemon juices obtained from the main Sicilian cultivars (Femminello comune, Monachello, and Interdonato). Significant amounts of an unusual constituent were found in the lemon juice of the above-mentioned cultivars together with eriocitrin, hesperidin, and diosmin. Following purification by preparative HPLC, the structure was assigned as 6,8-di-C-glucopyranosyldiosmetin by means of DAD-UV, ESI-MS-MS, and (1)H NMR analysis. Three other minor components were also detected. One of these presented a flavone nature, and spectral data and literature references both suggested a 6,8-di-C-glucopyranosylapigenin structure. The different contents of eriocitrin, hesperidin, diosmin, and above minor components in the cultivars allow juices to be readily differentiated.     

HL-60 differentiating activity and flavonoid content of the readily extractable fraction prepared from citrus juices

Citrus plants are rich sources of various bioactive flavonoids. To eliminate masking effects caused by hesperidin, naringin, and neoeriocitrin, the abundant flavonoid glycosides which make up 90% of the conventionally prepared sample, the readily extractable fraction from Citrus juice was prepared by adsorbing on HP-20 resin and eluting with EtOH and acetone from the resin and was subjected to HL-60 differentiation assay and quantitative analysis of major flavonoids. Screening of 34 Citrus juices indicated that King (C. nobilis) had a potent activity for inducing differentiation of HL-60, and the active principles were isolated and identified as four polymethoxylated flavonoids, namely, nobiletin, 3,3',4',5,6,7, 8-heptamethoxyflavone, natsudaidain, and tangeretin. HPLC analysis of the readily extractable fraction also indicated that King contained high amounts of these polymethoxylated flavonoids among the Citrus juices examined. Principal component and cluster analyses of the readily extractable flavonoids indicated peculiarities of King and Bergamot.     

Comparison of volatile concentrations in hand-squeezed juices of four different lemon varieties

Fresh juices of four Italian cultivars of lemons (Citrus limon Burm) have been analyzed by headspace solid phase microextraction coupled to gas chromatography and gas chromatography-mass spectrometry (GC-MS). The best results have been obtained with the 2 cm 50/30 microm divinylbenzene/carboxen on poly(dimethylsiloxane) fiber, using a homogenization time of 1 h at 40 degrees C and a sampling period of 30 min. A total of 35 volatile compounds have been identified by GC-MS, and their relative amounts have been calculated by adding internal standard to the samples. Differences in composition of lemon juices volatile components have been observed. Verdello Siracusano lemon juice has the highest amount of volatile compounds (50.28 mg/L), followed by Interdonato (8.39 mg/L), Primo Fiore Capo d'Orlando (5.75 mg/L), and Femminello Siracusano (2.62 mg/L) juices. Volatile compounds mainly consist of mono- and sesquiterpene hydrocarbons and oxygenated molecules (aldheydes, monoterpene alcohols, and monoterpene esters). Headspace solid phase microextraction coupled to a gas chromatograph equipped with a specific sulfur detector, a sulfur chemiluminescence detector, let us detect and quantify dimethyl sulfide compound at the microgram/liter level in lemon juices.     

Estimating bergamot juice adulteration of lemon juice by high-performance liquid chromatography (HPLC) analysis of flavanone glycosides

The chemical composition of 30 samples of juices obtained from bergamot (Citrus bergamia Risso and Poit.) fruits is reported and compared to the genuineness parameters adopted by Association of the Industry of Juice and Nectars (AIJN) for lemon juice. It was found that the compositional differences between the two juices are distinguishable, although with difficulty. However, these differences are not strong enough to detect the fraudulent addition of bergamot juice to lemon juice. Instead, we found the high-performance liquid chromatography (HPLC) analysis of the flavanones naringin, neohesperidin, and neoeriocitrin, which are present in bergamot juice and practically absent in the lemon juice, is a convenient way to detect and quantify the fraudulent addition of bergamot juice. The method has been validated by calculating the detection and quantification limits according to Eurachem procedures. Employing neoeriocitrin (detection limit = 0.7 mg/L) and naringin (detection limit = 1 mg/L) as markers, it is possible to detect the addition of bergamot juice to lemon juice at the 1% level. When using neohesperidin as a marker (detection limit = 1 mg/L), the minimal percentage of detectable addition of bergamot juice was about 2%. Finally, it is reported that the pattern of flavonoid content of the bergamot juice is similar to those of chinotto (Citrus myrtifolia Raf) and bitter orange (Citrus aurantium L.) juices and that it is possible to distinguish the three kinds of juices by HPLC analysis.     

Identification and antioxidant potential of flavonoids and low molecular weight phenols in olive cultivar chemlali growing in Tunisia

Increasing interest in phenolic compounds in olives is due to their antioxidant and health-enhancing properties. In this study the phenolics in fruits of the Tunisian olive cultivar Chemlali were extracted by methanol-water and fractionated using Sephadex LH-20 column chromatography. The identification of phenolic monomers and flavonoids was based on separation by high-performance liquid chromatography equipped with a diode array detector followed by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analysis. Oleuropein, a secoiridoid glycoside esterified with a phenolic acid, was the major compound. Eight phenolic monomers and 12 flavonoids were also identified in Chemlali olives. Five flavonoids were isolated and purified using Sephadex LH-20 column chromatography and preparative paper chromatography. The antioxidant activity of the extract and the purified compounds was evaluated by measuring the radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and by using the beta-carotene-linoleate model assay. Acid hydrolysis of the extract enhanced its antioxidant activity. Hydroxytyrosol and quercetin showed antioxidant activities similar to that of 2,6-di-tert-butyl-4-methylphenol. A hydroxyl group at the ortho position at 3' on the B ring of the flavonoid nucleus could contribute to the antioxidant activity of the flavonoids.     

Activity and concentration of polyphenolic antioxidants in apple juice. 2. Effect of novel production methods

There is a great interest in food components that possess possible health-protecting properties, as is the case with flavonoids. Previous research showed that conventional apple juice processing resulted in juices poor in flavonoids and with a low antioxidant activity. This paper shows that it is possible to improve flavonoid content in juice and its antioxidant activity by applying an alcoholic extraction either on the pulp or on the pomace. The levels of flavonoids and chlorogenic acid in enriched juice were between 1.4 (chlorogenic acid) and 9 (quercetin glycosides) times higher than in conventional apple juice. In enriched juice the antioxidant activity was 5 times higher than in conventional apple juice, with 52% of the antioxidant activity of the originating fruits present. The novel processing method had similar effects for three apple cultivars tested (Elstar, Golden Delicious, and Jonagold). The taste and color of enriched juice were different from those of conventional juice.     

Reliability of analytical methods for determining anthocyanins in blood orange juices

Quantitative analysis of anthocyanins was performed on a series of blood orange juices according to various spectrophotometric and HPLC methods, and the causes for different concentration resulting from the application of such procedures were investigated. Spectrophotometric methods utilizing aqueous ethanol as a solvent provided an anthocyanin content higher than that determined by HPLC. Discrepancies were ascribed to the use of impure standards and/or unsuitable calibration lines. The most consistent results with the HPLC findings were obtained by a method utilizing water as a solvent and cyanidin-3-glucoside as a standard. Actual concentration of anthocyanins in blood orange juice was remarkably lower than that currently determined by procedures used in the juice producing factories.     

Characterization of anthocyanins in grape juices by ion trap liquid chromatography-mass spectrometry

A reverse phase HPLC and electrospray interface with ion trap mass spectrometer method was developed for the characterization of anthocyanins in Concord, Rubired, and Salvador grape juices. Rubired and Salvador grapes are hybrids from Vitis vinifera and Vitis rupestris. Concord grape is a grape from the native American cultivar Vitis labrusca. Individual anthocyanins in these three varieties were identified on the basis of UV-vis and MS spectra and further elucidated by MS/MS spectra. Anthocyanins in Salvador and Concord grapes were 3-O-glucosides, 3-O-(6' '-O-p-coumaroyl)glucosides, 3-O-(6' '-O-p-acetyl)glucosides, 3,5-O-diglucosides, and 3-O-(6' '-O-p-coumaroyl)-5-O-diglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Vitisin B was detected in Salvador grape juice. Anthocyanins in Rubired grape juice were primarily anthocyanin diglucosides: peonidin 3,5-O-diglucoside, malvidin 3,5-O-diglucoside, peonidin 3-O-(6' '-O-p-coumaroyl)-5-O-diglucoside, and malvidin 3-O-(6' '-O-p-coumaroyl)-5-O-diglucoside are the four major anthocyanins. The presence of pelargonidin 3-O-glucoside, not previously reported, has been established for the first time in all three juices.     

Fresh Israeli Jaffa blond (Shamouti) orange and Israeli Jaffa red Star Ruby (Sunrise) grapefruit juices affect plasma lipid metabolism and antioxidant capacity in rats fed added cholesterol

The bioactivity of Israeli Jaffa blond (Shamouti) fresh orange and Israeli Jaffa red Star Ruby (Sunrise) grapefruit juices was investigated in vitro and in vivo. The contents of bioactive compounds of these juices were determined. The influence of bioactive compounds on plasma lipids and plasma antioxidant activity in rats fed cholesterol-containing and cholesterol-free diets was assessed. Significant differences in the contents of dietary fibers were not found. The contents of total polyphenols, flavonoids, and anthocyanins in fresh orange and grapefruit juices were 962.1 +/- 27.2 and 906.9 +/- 27.1; 50.1 +/- 3.3 and 44.8 +/- 3.2; and 69.9 +/- 5.6 and 68.7 +/- 5.5 microg/mL, respectively. The antioxidant potential measured by the scavenging activity against nitric oxide, the beta-carotene-linoleate model system (beta-carotene), and the 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diamonium salt assays was higher in orange juice but not significantly. A high level of correlation between contents of total polyphenols and flavonoids and antioxidant potential values of both juices was found. Diets supplemented with orange and to a lesser degree with grapefruit juices improved plasma lipid metabolism only in rats fed added cholesterol. However, an increase in the plasma antioxidant activity was observed in both groups. In conclusion, fresh orange and grapefruit juices contain high quantities of bioactive compounds, which guarantee their high antioxidant potential, and the positive influence on plasma lipid metabolism and plasma antioxidant activity could make fresh orange and grapefruit juices a valuable supplement for disease-preventing diets.     

Variation of flavonoids and furanocoumarins in grapefruit juices: a potential source of variability in grapefruit juice-drug interaction studies

Grapefruit juice (GFJ) has been found to interact with several medications, increasing their oral bioavailability and the risk of toxicity. Inhibition of CYP3A4 in the small intestine by flavonoids (such as naringin and naringenin) and furanocoumarins (including bergamottin and 6',7'-dihydroxybergamottin) present in GFJ seems to be the predominant mechanism, although P-glycoprotein and influx transporters in the small intestine are also involved. The quantity of interactive compounds ingested may affect the magnitude and mechanism of the food-drug interaction. Therefore, these four compounds were quantified by HPLC analysis in commercially available and fresh-squeezed GFJ and in grapefruit tissues. Considerable variability in naringin (174-1492 micromol/L), bergamottin (1.0-36.6 micromol/L), and 6',7'-dihydroxybergamottin (0.22-52.5 micromol/L) was observed, whereas naringenin could not be detected. White grapefruit showed higher concentrations of naringin and furanocoumarins located in the albedo and flavedo compared with red varieties. Findings from this study suggest considering concentrations of components with a potential for drug interactions in GFJ-drug interaction studies. The concentration of potentially contributing compounds may crucially influence the magnitude of observed interaction and impair direct comparison of studies in which different juices have been used.     

Analysis of bitter limonoids in citrus juices by atmospheric pressure chemical ionization and electrospray ionization liquid chromatography-mass spectrometry

Improved analytical techniques for bitter limonoids in citrus and citrus juices can expedite the evaluation of freeze-induced citrus damage for citrus growers and juice quality for citrus juice producers. Microbore normal-phase and reverse-phase chromatography coupled to a mass spectrometer operating in a positive ion atmospheric pressure chemical ionization and electrospray ionization modes were found to be rapid, selective, and sensitive methods for the analysis of the bitter limonoids limonin and nomilin in citrus juices. Analysis was performed on a chloroform extract of citrus juice to which an internal standard was added. The methods are capable of detecting citrus limonoids in citrus juice in the 60-200 picogram range and quantifying citrus juice limonoids in concentrations as low as 120 picograms. An accurate "total limonoid bitterness" in citrus juice, as represented by the combined occurrence of limonin and nomilin, is easily determined by these methods.     

Betaines in fruits of Citrus genus plants

Numerous compounds, many of them osmolytes, were quantified in natural juices and in frozen concentrate juices from fruits of plants of the Citrus genus. L-proline, N-methyl-L-proline (hygric acid), N,N-dimethyl-L-proline (stachydrine), 4-hydroxy-L-prolinebetaine (betonicine), 4-hydroxy-L-proline, γ-aminobutyric acid (Gaba), 3-carboxypropyltrimethylammonium (GabaBet), N-methylnicotinic acid (trigonelline), and choline in the fruit juices of yellow orange, blood orange, lemon, mandarin, bitter orange (Citrus aurantium), chinotto (Citrus myrtifolia), and grapefruit were analyzed by sensitive HPLC-ESI-tandem mass spectrometry procedure. It was found that the most represented osmolytes in the juices, that is, L-proline, stachydrine, and betonicine, can be quantified with minimal sample preparation and short analysis time (about 1 min) also by flow injection analysis (FIA) ESI-MS/MS with the same results as obtained by HPLC ESI-MS/MS. In all of the juices, discrete amounts of choline and trigonelline were present. Conversely, GabaBet was always below detection limits. Notably, N-methyl-L-proline and 4-hydroxy-L-prolinebetaine, which were discovered for the first time in the juice of bergamot (Citrus bergamia Risso et Poit), are also present in all of the citrus juices examined.     

Using an ion-trap MS sensor to differentiate and identify individual components in grapefruit juice headspace volatiles

An ion-trap mass spectrometer chemical sensor has been utilized to differentiate between grapefruit juices that differ only in the concentration of a single component, and the sensor was able to identify that component. Grapefruit juice was fortified with 40 to 2000 ppm vanillin, a low-level naturally occurring compound in citrus juices. Principal components analysis and discriminant analysis of mass spectral data (m/z 50-200) provided clear separation of the grapefruit juice samples. Vanillin was observed in the juice headspace at the 40 ppm level, with identification possible at the 100 ppm level using either MS or MS/MS.     

Characterization of anthocyanins and pyranoanthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juice

Anthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juices were isolated and purified by means of high-speed countercurrent chromatography and preparative high-performance liquid chromatography. Structures of the pigments were then elucidated by electrospray ionization multiple mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The major anthocyanins of the juice were characterized as cyanidin 3-glucoside and cyanidin 3-(6"-malonylglucoside). Furthermore, six minor anthocyanins were detected and identified as cyanidin 3,5-diglucoside, delphinidin 3-glucoside, cyanidin 3-sophoroside, delphinidin 3-(6"-malonylglucoside), peonidin 3-(6"-malonylglucoside), and cyanidin 3-(6"-dioxalylglucoside). The occurrence of the latter compound in blood oranges is reported here for the first time, together with full NMR spectroscopic data. Further investigations revealed the presence of four anthocyanin-derived pigments, which are formed through a direct reaction between anthocyanins and hydroxycinnamic acids during prolonged storage of the juice. These novel pyranoanthocyanins were identified as the 4-vinylphenol, 4-vinylcatechol, 4-vinylguaiacol, and 4-vinylsyringol adducts of cyanidin 3-glucoside through comparison of their mass spectrometric and chromatographic properties with those of synthesized reference compounds.     

Ellagic acid and flavonoid antioxidant content of muscadine wine and juice

Antioxidant properties of flavonoids and ellagic acid were characterized in eight wines and juices produced by various processing methodologies from red and white muscadine grape cultivars (Vitis rotundifolia). Juices and wines were produced by hot- and cold-pressed techniques, and additional wine was produced following on-hull fermentation for 3, 5, and 7 days. Chromatographic conditions were developed to simultaneously separate anthocyanins, ellagic acid, and flavonols and correlated to a measurement of overall antioxidant capacity (AOX), and their changes were monitored after storage for 60 days at 20 and 37 degrees C. Regression coefficients between concentrations of individual polyphenolics and AOX ranged from 0.55 for ellagic acid to 0.90 for kaempferol. Both red and white wines had higher AOX values after storage than juices made from an identical grape press, despite lower concentrations of individual polyphenolic compounds. Red wines fermented on-hull had higher initial concentrations of antioxidant polyphenolics as compared to a corresponding hot-pressed juice, but changes in AOX during storage were more affected by time than by storage temperature despite lower concentrations of flavonoids and ellagic acid present at 37 degrees C as compared to 20 degrees C. Oxidative or polymerization reactions significantly decreased levels of monomeric anthocyanins during storage with the greatest losses observed for delphinidin and petunidin 3,5-diglucosides. Processing methods for muscadine wine and juice production were important factors influencing concentrations of antioxidant flavonoids and ellagic acid, while the role of fermentation and time had the greatest influence on retention of AOX properties during storage.     

Investigation of different apple cultivars by high resolution magic angle spinning NMR. A feasibility study

(1)H HR-MAS NMR spectroscopy was applied to apple tissue samples deriving from 3 different cultivars. The NMR data were statistically evaluated by analysis of variance (ANOVA), principal component analysis (PCA), and partial least-squares-discriminant analysis (PLS-DA). The intra-apple variability of the compounds was found to be significantly lower than the inter-apple variability within one cultivar. A clear separation of the three different apple cultivars could be obtained by multivariate analysis. Direct comparison of the NMR spectra obtained from apple tissue (with HR-MAS) and juice (with liquid-state HR NMR) showed distinct differences in some metabolites, which are probably due to changes induced by juice preparation. This preliminary study demonstrates the feasibility of (1)H HR-MAS NMR in combination with multivariate analysis as a tool for future chemometric studies applied to intact fruit tissues, e.g. for investigating compositional changes due to physiological disorders, specific growth or storage conditions.     

Multielement analysis of argentinean lemon juices by instrumental neutronic activation analysis and their classification according to geographical origin

Multielement analysis of lemon juices from different Argentinean regions was carried out by instrumental neutronic activation analysis (INAA) with the aim at developing a reliable method in the traceability of the origin of lemon juices. This work presents a characterization of 44 lemon juice samples selected from three different geographical origins in the Northwest region of Argentina. Multivariate chemometric techniques such principal component analysis and lineal discriminant analysis (LDA) were used with the aim of classifying the juices and identifying the most significant parameters. Eleven elements were determined (Br, As, Na, Rb, La, Cr, Sc, Fe, Co, Zn, and Sb). The analytical method was validated by analyzing the standard reference material IAEA V-10 (hay powder); the results are within +/-10% of the reported values for the majority of the elements. Biplots of LDA scores for the INAA data illustrate clear separation between each sample.     

Orange, mandarin, and hybrid classification using multivariate statistics based on carotenoid profiles

A study was undertaken to differentiate citrus on the basis of a carotenoid profile obtained from the HPLC determination of 12 carotenoids found in saponified fresh juice. Mandarin, orange, and various hybrid varieties were analyzed to determine their carotenoid profiles. The resulting peak areas were analyzed using principal component analysis (PCA), canonical discriminate analysis (CDA), and Mahalanobis distances. These were used to develop models for classifying the juices into appropriate groups. Thirty-two samples were analyzed to determine classification techniques. Mandarin and orange juices were quite distinct, with the hybrids scattered throughout the mandarin and orange clusters using PCA. CDA resulted in three distinct groups with only a few of the hybrids in the orange grouping.     

锦橙汁辐照和巴氏灭菌处理后相关品质的分析

为探讨辐照和巴氏杀菌对橙汁品质的影响,对锦橙汁分别进行1.4、2.8、5.6kGy3种不同剂量辐照及巴氏灭菌处理,使用固相微萃取-气质联用技术对挥发性成分进行分析,测定橙汁色度、pH值和Vc含量,并对橙汁香气进行感官评定。在鲜橙汁、巴氏灭菌汁和3种辐照样品中分别检测到了54、47、57、55和53种成分,2.8kGy剂量辐照处理后橙汁挥发性成分总峰面积最高,β-月桂烯、柠檬烯和γ-松油烯等橙汁特征香气物质经辐照后保持率高于巴氏灭菌。各种处理后Vc含量均有所下降,橙汁橙香均减弱。1.4kGy辐照后的橙汁感官评价结果最好,因此可以对橙汁进行低剂量辐照灭菌。