Quantitative high-performance liquid chromatography analyses of flavonoids in Australian Eucalyptus honeys

Flavonoids of nine Australian monofloral Eucalyptus honeys have been analyzed and related to their botanical origins. The mean content of total flavonoids varied from 1.90 mg/100 g of honey for stringybark (E. globoidia) honey to 8.15 mg/100 g of honey for narrow-leaved ironbark (E. crebra) honey, suggesting that species-specific differences occur quantitatively among these Eucalyptus honeys. All of the honey samples analyzed in this study have a common flavonoid profile comprising tricetin (5,7,3',4',5'-pentahydroxyflavone), quercetin (3,5,7,3',4'-pentahydroxyflavone), and luteolin (5,7,3',4'-tetrahydroxyflavone), which, together with myricetin (3,5,7,3',4',5'-hexahydroxyflavone) and kaempferol (3,5,7,4'-tetrahydroxyflavone), were previously suggested as floral markers for European Eucalyptus honeys. Thus, flavonoid analysis could be used as an objective method for the authentication of the botanical origin of Eucalyptus honeys. Moreover, species-specific differences can also be found in the composition of honey flavonoid profiles. Among these honeys, bloodwood (E. intermedia) honey contains myricetin and tricetin as the main flavonoid compounds, whereas there is no myricetin detected in yapunyah (E. ochrophloia), narrow-leaved ironbark (E. crebra), and black box (E. largiflorens) honeys. Instead, these types of Eucalyptus honeys may contain tricetin, quercetin, and/or luteolin as their main flavonoid compounds. Compared to honeys from other geographical origins, the absence or minor presence of propolis-derived flavonoids such as pinobanksin, pinocembrin, and chrysin in Australian honeys is significant. In conclusion, these results demonstrate that a common flavonoid profile exists for all of the Eucalyptus honeys, regardless of their geographical origins; the individual species-specific floral types of Eucalyptus honey so common in Australia could be possibly differentiated by their flavonoid profile differences, either qualitatively or quantitatively or both.     

Floral classification of honey using mid-infrared spectroscopy and surface acoustic wave based z-Nose Sensor

Fourier transform infrared spectroscopy (FTIR) and z-Nose were used as screening tools for the identification and classification of honey from different floral sources. Honey samples were scanned using microattenuated total reflectance spectroscopy in the region of 600-4000 cm(-1). Spectral data were analyzed by principal component analysis, canonical variate analysis, and artificial neural network for classification of the different honey samples from a range of floral sources. Classification accuracy near 100% was achieved for clover (South Dakota), buckwheat (Missouri), basswood (New York), wildflower (Pennsylvania), orange blossom (California), carrot (Louisiana), and alfalfa (California) honey. The same honey samples were also analyzed using a surface acoustic wave based z-Nose technology via a chromatogram and a spectral approach, corrected for time shift and baseline shifts. On the basis of the volatile components of honey, the seven different floral honeys previously mentioned were successfully discriminated using the z-Nose approach. Classification models for FTIR and z-Nose were successfully validated (near 100% correct classification) using 20 samples of unknown honey from various floral sources. The developed FTIR and z-Nose methods were able to detect the floral origin of the seven different honey samples within 2-3 min based on the developed calibrations.     

Discrimination and classification of olive tree varieties and cultivation zones by biophenol contents

The peak areas from a high-performance liquid chromatography-diode array (HPLC-DAD) analysis of biophenols extracted from olive leaves have been used as chemotaxonomic markers to construct chemometric models in order to discriminate and classify (1) 13 varieties of Olea europaea olive trees, namely, Alame?o, Arbequina, Azulillo, Chorna, Hojiblanca, Lechín, Manzanillo, Negrillo, Nevadillo, Ocal, Pierra, Sevillano, and Tempranillo, from the same cultivation zone and (2) Arbequina samples from six different geoghaphical origins, namely, Córdoba, Mallorca (north and south), Ciudad Real, Lleida, and Navarra. Models based on principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used for discrimination between samples as a function of the tree varieties and cultivation zone, whereas K nearest neighbors (KNN) and soft independent modeling of class analogy (SIMCA) models were generated to classify the samples used to validate the models into one of the groups previously established by PCA and HCA. KNN classified correctly 93 and 92% of the samples into the variety and cultivation zone, respectively; meanwhile, the SIMCA models predicted 85 and 92%, respectively.     

Nectar Flavonol rhamnosides are floral markers of acacia (Robinia pseudacacia) honey

With the objective of finding floral markers for the determination of the botanical origin of acacia (robinia) honey, the phytochemicals present in nectar collected from Robinia pseudacacia flowers were analyzed by high-performance liquid chromatography-tandem mass spectrometry. Eight flavonoid glycosides were detected and characterized as kaempferol combinations with rhamnose and hexose. Acacia honey produced in the same location where the nectar was collected contained nectar-derived kaempferol rhamnosides. This is the first time that flavonoid glycosides have been found as honey constituents. Differences in the stability of nectar flavonoids during honey elaboration and ripening in the hive were shown to be due to hydrolytic enzymatic activity and to oxidation probably related to hydrogen peroxide (glucose-oxidase) activity. Acacia honeys contained propolis-derived flavonoid aglycones (468-4348 microg/100 g) and hydroxycinnamic acid derivatives (281-3249 microg/100 g). In addition, nectar-derived kaempferol glycosides were detected in all of the acacia honey samples analyzed (100-800 microg/100 g). These flavonoids were not detected in any of the different honey samples analyzed previously from different floral origins other than acacia. Finding flavonoid glycosides in honey related to floral origin is particularly relevant as it considerably enlarges the number of possible suitable markers to be used for the determination of the floral origin of honeys.     

Identification of botanical biomarkers in Argentinean Diplotaxis honeys: flavonoids and glucosinolates

To select and establish floral biomarkers of the botanical origin of Diplotaxis tenuifolia honeys, the flavonoids and glucosinolates present in bee-deposited nectar collected from hive combs (unripe honey) and mature honey from the same hives fron which the unripe honey samples were collected were analyzed by LC-UV-PAD-ESI-MS(n). Glycosidic conjugates of the flavonols quercetin, kaempferol, and isorhamnetin were detected and characterized in unripe honey. D. tenuifolia mature honeys contained the aglycones kaempferol, quercetin, and isorhamnetin. The differences between the phenolic profiles of mature honey and freshly deposited honey could be due to hydrolytic enzymatic activities. Aliphatic and indole glucososinolates were analyzed in unripe and mature honeys, this being the first report of the detection and characterization of glucosinolates as honey constituents. Moreover, these honey samples contained different amounts of propolis-derived flavonoid aglycones (1765-3171 μg/100 g) and hydroxycinnamic acid derivatives (29-1514 μg/100 g). Propolis flavonoids were already present in the freshly deposited nectar, showing that the incorporation of these compounds to honey occurs at the early steps of honey production. The flavonoids quercetin, kaempferol, and isorhamnetin and the glucosinolates detected in the samples could be used as complementary biomarkers for the determination of the floral origin of Argentinean Diplotaxis honeys.     

Characterization of the most odor-active volatiles of orange wine made from a Turkish cv. Kozan (Citrus sinensis L. Osbeck)

The aroma-active compounds of cv. Turkish Kozan orange wine were analyzed by sensory and instrumental analyses. Liquid-liquid extraction with dichloromethane was used for extraction of volatile components. According to sensory analysis, the aromatic extract obtained by liquid-liquid extraction was representative of orange wine odor. A total of 63 compounds were identified and quantified in orange wine. The results of the gas chromatography-olfactometry analysis showed that 35 odorous compounds were detected by the panelists. Of these, 28 aroma-active compounds were identified. Alcohols followed by terpenes and esters were the most abundant aroma-active compounds of the orange wine. Among these compounds, ethyl butanoate (fruity sweet), 3-methyl-1-pentanol (roasty), linalool (floral citrusy), gamma-butyrolactone (cheesy burnt sugar), 3-(methylthio)-propanol (boiled potato, rubber), geraniol (floral citrusy), and 2-phenylethanol (floral rose) were the most important contributors to the aroma of the orange wine because they were perceived by all eight panelists.     

Analysis of volatiles from Spanish honeys by solid-phase microextraction and gas chromatography-mass spectrometry

Headspace solid-phase microextraction (SPME), followed by gas chromatography (GC)-mass spectrometry (MS) determination, has been used for the analysis of honey volatiles. Two SPME fibers were employed to study the composition of volatiles from various types of Spanish honeys. The best results were obtained with the Carboxen/PDMS fiber, using a homogenization time of 1 h at 70 degrees C and a sampling period of 30 min. A total of 35 compounds were detected, most of them identified by GC-MS and quantified using external standards. Differences in the composition of honey volatiles were obtained, and these results allowed the differentiation of honeys. However, further studies are necessary to confirm the utility of this technique as an alternative tool for the characterization of the floral origin of honeys.     

The use of carbohydrate profiles and chemometrics in the characterization of natural honeys of identical geographical origin

A study of the real possibilities of carbohydrate profiles and chemometrics to characterize the botanical origin of honey from a single geographical area, the Province of Soria (Spain), is presented. To this end, 14 carbohydrates were quantified using high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD) in 77 natural honeys, the botanical origins of which were ling, spike lavender, French lavender, thyme, forest, and multifloral. Principal component analysis has been employed as a first approach to characterize the honey samples analyzed, showing similarities between spike lavender and multifloral honeys. The best discrimination among groups is obtained when four canonical discriminant analyses were carried out sequentially, origin by origin, achieving an overall percentage of success of 90% following cross-validation.     

Identification and quantification of antioxidant components of honeys from various floral sources

Little is known about the individual components of honey that are responsible for its antioxidant activity. The present study was carried out to characterize the phenolics and other antioxidants present in honeys from seven floral sources. Chromatograms of the phenolic nonpolar fraction of the honeys indicated that most honeys have similar but quantitatively different phenolic profiles. Many of the flavonoids and phenolic acids identified have been previously described as potent antioxidants. A linear correlation between phenolic content and ORAC activity was demonstrated (R(2) = 0.963, p < 0.0001). Honeys were separated by solid-phase extraction into four fractions for sugar removal and separation based on solubility to identify the relative contribution of each fraction to the antioxidant activity of honey. Antioxidant analysis of the different honey fractions suggested that the water-soluble fraction contained most of the antioxidant components. Specific water-soluble antioxidant components were quantified, including protein; gluconic acid; ascorbic acid; hydroxymethylfuraldehyde; and the combined activities of the enzymes glucose oxidase, catalase and peroxidase. Of these components, a significant correlation could be established only between protein content and ORAC activity (R(2) = 0.674, p = 0.024). In general, the antioxidant capacity of honey appeared to be a result of the combined activity of a wide range of compounds including phenolics, peptides, organic acids, enzymes, Maillard reaction products, and possibly other minor components. The phenolic compounds contributed significantly to the antioxidant capacity of honey but were not solely responsible for it.     

Analysis of phenolic and other aromatic compounds in honeys by solid-phase microextraction followed by gas chromatography-mass spectrometry

The solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS) was used for the analysis of phenolic and other aromatic compounds in honey samples from different floral origin. Different parameters affecting the efficiency of the extraction, such as the type of the stationary phase of the fiber, NaCl and acetic acid addition, and extraction time, were optimized for the detection of the maximum number of compounds in the shortest analysis time. A total of 31 compounds were detected, with most of them identified and quantified by GC-MS. The principal component analysis (PCA) was applied to the data matrix; the results allowed for the differentiation between honeydew and nectar honeys on the basis of the salicylic acid concentration. It was found that this acid has a high contribution in the honeydew group (71.2-705.9 microg/100 g of honey) compared to the nectar honey group (0-47.6 microg/100 g of honey). The comparison of data in each honey group enabled us to characterize the floral source of some honeys using some aromatic compounds as markers.     

Classification of Italian honeys by 2D HR-NMR

The importance of honey has been recently increased because of its nutrient and therapeutic effects, but the adulteration of honey in terms of botanical origin has increased, too. The floral origin of honeys is usually determined using melisso-palynological analysis and organoleptic characteristics, but the application of these techniques requires some expertise. A number of papers have confirmed the possibility of characterizing honey samples by selected chemical parameters. In this study high-resolution nuclear magnetic resonance (HR-NMR) and multivariate statistical analysis methods were used to identify and classify honeys of five different floral sources. The 71 honey samples (robinia, chestnut, citrus, eucalyptus, polyfloral) were analyzed by HR-NMR using both 1H NMR and heteronuclear multiple bond correlation spectroscopy (HMBC). Spectral data were analyzed by application of unsupervised and supervised pattern recognition and multivariate statistical techniques such as principal component analysis (PCA) and general discriminant analysis (GDA). The use of 1H-(13)C HMBC coupled with appropriate statistical analysis seems to be an efficient technique for the classification of honeys.     

Honey characterization and adulteration detection by pattern recognition applied on HPAEC-PAD profiles. 1. Honey floral species characterization

An improved COFRAC (COmité FRan?ais d'ACréditation) method for the analysis and evaluation of the quality of honey by high-performance anion-exchange chromatography of sugar profiles is proposed. With this method, both minor and major sugars are simultaneously analyzed and the technique is integrated in a new chemometric approach, which uses the entire chromatographic sugars profile of each analyzed sample to characterize honey floral species. Sixty-eight authentic honey samples (6 varieties) were analyzed by high-performance anion-exchange chromatography-pulsed amperometric detection. A new algorithm was developed to create automatically the corresponding normalized data matrix, ready-to-use in various chemometric procedures. This algorithm transforms the analytical profiles to produce the corresponding calibrated table of the surfaces or intensities according to retention times of peaks. The possibility of taking into account unknown peaks (those for which no standards are available) allows the maximum chemical information provided by the chromatograms to be retained. The parallel application of principal component analysis (PCA)/linear discriminant analysis (LDA) and artificial neural networks (ANN) shows a high capability in the classification of the analyzed samples (LDA, 93%; ANN, 100%) and a very good discrimination of honey groups. This work is the starting point of the elaboration of a new system designed for the automatic pattern recognition of food samples (first application on honey samples) from chromatographic analyses for food characterization and adulteration detection.     

Influence of storage conditions on chemical composition and sensory properties of citrus honey

Fresh citrus honey was stored at 10, 20, and 40 degrees C for 12 months. The effect of storage on the quality of honey was evaluated using physicochemical parameters, volatile compounds, mono-, di-, and trisaccharides, and sensory analysis. Diastase activity and HMF were out of the legal limit in honey stored 12 months at 40 degrees C. Volatile compounds (especially terpenes and terpene derivatives), monosaccharides, and disaccharides presented important losses during honey storage at any temperature. Honey storage at 10 or 20 degrees C maintained their floral, fresh, citric, and fresh fruit aroma, while the intensities of these attributes were diminished. Storage at 40 degrees C during 12 months resulted in the appearance of attributes such as "medicinal, smoked, toasted, cooked vegetable, and ripened fruit", associated with compounds formed during the Maillard reaction or through degradation of sugars such as volatile pyrroles, furanones, pyranones, and pyrazines, which appeared or increased in concentration during honey storage mainly at high temperature.     

Flavonoids in monospecific eucalyptus honeys from Australia

The HPLC analyses of Australian unifloral Eucalyptus honeys have shown that the flavonoids myricetin (3,5,7,3',4', 5'-hexahydroxyflavone), tricetin (5,7,3',4',5'-pentahydroxyflavone), quercetin (3,5,7,3',4'-pentahydroxyflavone), luteolin (5,7,3', 4'-tetrahydroxyflavone), and kaempferol (3,5,7, 4'-tetrahydroxyflavone) are present in all samples. These compounds were previously suggested as floral markers of European Eucalyptus honeys. The present results confirm the use of flavonoid analysis as an objective method for the botanical origin determination of eucalyptus honey. Honeys from E. camaldulensis (river red gum honey) contain tricetin as the main flavonoid marker, whereas in honeys from E. pilligaensis (mallee honey), luteolin is the main flavonoid marker, suggesting that species-specific differences can be detected with this analysis. The main difference between the flavonoid profiles of Australian and European Eucalyptus honeys is that in the Australian honeys, the propolis-derived flavonoids (pinobanksin (3,5, 7-trihydroxyflavanone), pinocembrin (5,7-dihydroxyflavanone), and chrysin (5,7-dihydroxyflavone)) are seldom found and in much smaller amounts.     

Assessment of the floral origin of honey by SDS-page immunoblot techniques

We report on the development of a novel alternative method for the assessment of floral origin in honey samples based on the study of honey proteins using immunoblot assays. The main goal of our work was to evaluate the use of honey proteins as chemical markers of the floral origin of honey. Considering that honeybee proteins should be common to all types of honey, we decided to verify the usefulness of pollen proteins as floral origin markers in honey. We used polyclonal anti-pollen antibodies raised in rabbits by repeated immunization of Sunflower (Elianthus annuus) and Eucalyptus (Eucalyptus sp.) pollen extracts. The IgG fraction was purified by immunoaffinity. These antibodies were verified with nitrocellulose blotted pollen and unifloral honey protein extracts. The antibodies anti-Sunflower pollen, bound to the 36 and 33 kDa proteins of Sunflower unifloral honey and to honey containing Sunflower pollen; and the antibodies anti-Eucalyptus sp. pollen bound to the 38 kDa proteins of Eucalyptus sp. unifloral honey in immunoblot assays. Satisfactory results were obtained in differentiating between the types of pollen analyzed and between Sunflower honey and Eucalyptus honey with less cross reactivity with other types of honey from different origin and also with good sensitivity in the detection. This immunoblot method opens an interesting field for the development of new antibodies from different plants, which could serve as an alternative or complementary method to the usual melissopalynological analysis to assess honey floral origin.     

Characterization of aroma compounds responsible for the rosy/floral flavor in Cheddar cheese

The aroma-active compounds that contribute to the rosy/floral flavor in Cheddar cheese were characterized using both instrumental and sensory techniques. Two cheeses (>12 months old) with rosy/floral flavor and two Cheddar cheeses of similar ages without rosy/floral flavors were selected. After direct solvent extraction/solvent-assisted flavor evaporation and separation into neutral/basic and acidic fractions, samples were analyzed by gas chromatography-olfactometry with aroma extract dilution analysis. Selected compounds were quantified using internal standard methodology. Some of the intense aroma-active compounds in the neutral basic fraction of the rosy/floral cheeses included 2-phenethanol (rosy), phenylethyl acetate (rosy), and phenylacetaldehyde (rosy/floral). Quantification, threshold analysis, and sensory analysis of model cheeses confirmed that increased concentrations of phenylacetaldehyde and phenylacetic acid caused rosy/floral flavor when spiked into Cheddar cheese.     

Identification of flavonoid markers for the botanical origin of Eucalyptus honey

European Eucalyptus honeys showed a common and characteristic HPLC profile in which the flavonoids myricetin (3,5,7,3',4', 5'-hexahydroxyflavone), tricetin (5,7,3',4',5'-pentahydroxyflavone), quercetin (3,5,7,3',4'-pentahydroxyflavone), luteolin (5,7,3', 4'-tetrahydroxyflavone), and kaempferol (3,5,7, 4'-tetrahydroxyflavone) were identified. Their contents, and relative amounts, in the analyzed honey samples were quite constant and supported their floral origin. In addition, ellagic acid and the propolis-derived flavonoids pinobanksin, pinocembrin, and chrysin were detected in most samples. The contents of these nonfloral phenolics were much more variable as could be expected for their propolis origin. Myricetin, tricetin, and luteolin had not been identified as floral markers in any other honey sample previously analyzed in our laboratory (chestnut, citrus, rosemary, lavender, acacia, rapeseed, sunflower, heather, lime tree, etc.) or reported in the literature, suggesting that these could be useful markers. Only in some individual heather samples produced in Portugal has tricetin previously been detected in minor amounts. These samples, however, were contaminated with Eucalyptus as revealed by their pollen analysis and the lack of tricetin or their glycosides in heather floral nectar. It remains to be established if myricetin, tricetin, and luteolin originate from Eucalyptus floral nectar where the corresponding glycosides should be present.     

Soil volatile analysis by proton transfer reaction-time of flight mass spectrometry (PTR-TOF-MS)

We analyzed the volatile organic compounds (VOCs) emitted from different soils by using the PTR-MS-TOF technique under laboratory conditions and compared them with soil chemical biochemical activities. The emitted VOCs were related to soil microbial biomass, soil respiration and some soil enzyme activities so as to evaluate if size and activity of soil microbial communities influenced the soil VOCs profiles. Our results showed that the emitted VOCs discriminated between soils with different properties and management, and differences in the VOCs emission profiles were likely related to the active metabolic pathways in the microbial communities of the three studied soil. Our results also showed that some soil enzyme activities such as β-glucosidase and arylsulfatase were possibly involved in the release of compounds fueling microbial metabolic pathways leading to the production of specific VOCs. It was concluded that the PTR-MS-TOF technique is suitable for analyze VOCs emission from soil and that studies comparing soil enzyme activities and soil volatile profiles can reveal the origin of VOCs and give further insights on microbial activity and soil functionality.     

Lumichrome and phenyllactic acid as chemical markers of thistle (Galactites tomentosa Moench) honey

HPLC-DAD-MS/MS chromatograms of thistle (Galactites tomentosa Moench) unifloral honeys, previously selected by sensory evaluation and melissopalynological analysis, showed high levels of two compounds. One was characterized as phenyllactic acid, a common acid found in honeys, but the other compound was very unusual for honeys. This compound was extracted from honey with ethyl acetate and purified by SPE using C(18), SiOH, and NH(2) phases. Its structure was elucidated on the basis of extensive 1D and 2D NMR experiments as well as HPLC-MS/MS and Q-TOF analysis, and it was identified as lumichrome (7,8-dimethylalloxazine). Lumichrome is known to be the main product of degradation obtained in acid medium from riboflavin (vitamin B(2)), and this is the first report of the presence of lumichrome in honeys. Analysis of the G. tomentosa raw honey and flowers extracts confirmed the floral origin of this compound. The average amount of lumichrome in thistle honey was 29.4 ± 14.9 mg/kg, while phenyllactic acid was 418.6 ± 168.9 mg/kg. Lumichrome, along with the unusual high level of phenyllactic acid, could be used as a marker for the botanical classification of unifloral thistle (G. tomentosa) honey.     

Preliminary chemometric study on the use of honey as an environmental marker in Galicia (northwestern Spain)

Thirteen metal elements were determined in 40 honey samples from Galicia with different environmental origins: rural, urban, and industrial areas. The data set of the honey metallic profiles was studied with a double purpose: first, to make a preliminary evaluation of honey as an environmental indicator in Galicia with the aim of monitoring pollution and, second, to compare the different capabilities of diverse pattern recognition prediction procedures for modeling the environmental surrounding of the hive. A certain level of similarity for urban and industrial samples was obtained using principal component analysis and cluster analysis, whereas significant differences for urban and industrial honeys were found in relation to rural honey samples. Different classification rules to associate metal content of honeys with their environmental surrounding were obtained by chemometric pattern recognition procedures. In general, the classification methods developed by neural networks provided better results than the traditional pattern recognition procedures. The metal profiles of honey seem to provide sufficient information to enable categorization criteria for classifying samples according to their environmental surrounding. Thus, honey could be a potential pollution indicator for the Galician area.