Ignazio Floris

RP-HPLC determination of water-soluble vitamins in honey

The assessment and validation of reliable analytical methods for the determination of vitamins in sugar-based matrices (e.g. honey) are still scarcely explored fields of research. This study proposes and fully validates a simple and fast RP-HPLC method for the simultaneous determination of five water-soluble vitamins (vitamin B(2), riboflavin; vitamin B(3), nicotinic acid; vitamin B(5), pantothenic acid; vitamin B(9), folic acid; and vitamin C, ascorbic acid) in honey. The method provides low detection and quantification limits, very good linearity in a large concentration interval, very good precision, and the absence of any bias. It has been successfully applied to 28 honey samples (mainly from Sardinia, Italy) of 12 different botanical origins. While the overall amount of the analytes in the samples is quite low (always below 40 mg kg(-1)), we have observed a marked dependence of some of their concentrations (i.e. vitamin B(3) and vitamin B(5)) and the botanical origin of the honey. This insight might lead to important characterization features for this food item.

LD50 and Repellent Effects of Essential Oils from Argentinian Wild Plant Species on Varroa destructor

Abstract The repellent and acaricidal effects of some essential oils from the most typical wild plant species of northern Patagonia, Argentina, on Varroa destructor Anderson & Trueman were evaluated using a complete exposure test. Honey bees, Apis mellifera L., and mites (five specimens of each per dish) were introduced in petri dishes having different oil concentrations (from 0.1 to 25 μl per cage). Survival of bees and mites was registered after 24, 48, and 72 h. An attraction/repellence test was performed using a wax tube impregnated with essential oil and another tube containing wax only. The lowest LD50 values for mites were registered for Acantholippia seriphioides (A. Gray) Mold. (1.27 μl per cage) and Schinus molle L. (2.65 μl per cage) after 24 h, and for Wedelia glauca (Ortega) O. Hoffm. ex Hicken (0.59 μl per cage) and A. seriphioides (1.09 μl per cage) after 72 h of treatment. The oil with the highest selectivity ratio (A. mellifera LD50/V. destructor LD50) was the one extracted from S. molle (>16). Oils of Lippia junelliana (Mold.) Troncoso, Minthostachys mollis (HBK) Grieseb., and Lippia turbinata Grieseb. mixed with wax had repellent properties. None of the oils tested had attractive effects on Varroa mites.

Methyl Syringate: A Chemical Marker of Asphodel (Asphodelus microcarpus Salzm. et Viv.) Monofloral Honey

During the liquid chromatographic study of the phenolic fraction of monofloral honeys was detected in the asphodel honey ( Asphodelus microcarpus Salzm. et Viv.) chromatogram a distinctive peak not detected in other monofloral honeys such as Arbutus unedo L., Hedysarum coronarium , Eucalyptus spp., and Galactites tomentosa . After thin layer chromatography (TLC) purification and characterization by NMR and LC-MS/MS, the compound was identified as methyl syringate (MSYR) and confirmed against an original standard. Levels of MSYR were measured in honeys of 2005, 2006, and 2007 by HPLC-DAD analysis. Level determination of MSYR was repeated in 2008 for 2006 and 2007 honeys to evaluate chemical stability of this phenolic compound. Levels of MSYR measured 1 year after the sampling did not show significant statistical differences (p < 0.05). The stability of MSYR was also confirmed by 12 asphodel honey samples collected in 2005 that showed amounts of methyl syringate comparable with those found in fresh honey. For the evaluation of MSYR origin, samples of nectars were collected from flowers and the content of MSYR was measured. Levels of MSYR in honeys are originated from the nectar with an average contribution of the nectar to the honey of 80%. Melissopalinological analysis did not allow the attribution of the honey monofloral origin because levels of asphodel pollen were <6% for all analyzed samples. Previously reported levels of MSYR for robinia, rape, chestnut, clover, linden blossom, dandelion, sunflower, thyme, manuka, and fir honeys were <5 mg/kg. For this reason, a minimum level of 122.6 mg/kg for MSYR in asphodel honeys can be considered as a chemical marker and, unlike the melissopalynological analysis, can be used for the origin attribution and to evaluate the percent of asphodel nectar in the honey.

A direct RP-HPLC method for the determination of furanic aldehydes and acids in honey.

In this study 5-hydroxymethyl-2-furaldehyde (HMF), 2-furaldehyde, 3-furaldehyde, 2-furoic acid and 3-furoic acid are contemporarily determined in honey using a swift and direct RP-HPLC approach. The validation protocol was performed in terms of detection and quantification limits, precision (by repeatability and reproducibility), linearity and accuracy (by recovery tests); the acceptability of the precision and accuracy results was positively verified using Horwitzs model and AOAC guidelines, respectively. The method was tested on 18 honey samples of different ages, and botanical and geographical origin. HMF and 2-furaldehyde correlated highly with the age of the samples, whereas no correlation was observed with regards to 2-furaldehyde and 2-furoic acid. Hypotheses relating to the formation of minority furanic compounds are also proposed.

Floral Markers of Strawberry Tree (Arbutus unedo L.) Honey

Strawberry tree honey, due to its characteristic bitter taste, is one of the most typical Mediterranean honeys, with Sardinia being one of the largest producers. According to specific chemical studies, homogentisic acid was identified as a possible marker of this honey. This work, based on HPLC-DAD-MS/MS analysis of strawberry tree (Arbutus unedo L.) honeys, previously selected by sensory evaluation and melissopalynological analysis, showed that, in addition to the above-mentioned acid, there were other high levels of substances useful for the botanical classification of this unifloral honey. Two of these compounds were isolated and identified as (+/-)-2-cis,4-trans-abscisic acid (c,t-ABA) and (+/-)-2-trans,4-trans-abscisic acid (t,t-ABA). A third compound, a new natural product named unedone, was characterized as an epoxidic derivative of the above-mentioned acids. Structures of c,t-ABA, t,t-ABA, and unedone were elucidated on the basis of extensive 1D and 2D NMR experiments, as well as HPLC-MS/MS and Q-TOF analysis. In selected honeys the average amounts of c,t-ABA, t,t-ABA, and unedone were 176.2+/-25.4, 162.3+/-21.1, and 32.9+/-7.1 mg/kg, respectively. Analysis of the A. unedo nectar confirmed the floral origin of these compounds found in the honey. Abscisic acids were found in other unifloral honeys but not in such high amount and with a constant ratio of about 1:1. For this reason, besides homogentisic acid, these compounds could be used as complementary markers of strawberry tree honey.

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.

Lethal and sublethal effects of Brevibacillus laterosporus on the housefly ( Musca domestica )

Sporulated cultures of an isolate of the entomopathogenic bacterium Brevibacillus laterosporus (Laubach) from soil in Sardinia (Italy) were highly toxic by ingestion to both adults and juveniles of the housefly, Musca domestica L. (Diptera: Muscidae). Toxicity was associated with spores as the culture supernatant was not toxic and no parasporal inclusions were observed. The level of toxicity varied with the concentration of spores, the time of exposure to the treated diet, and the stage of insect development. Comparative LC50 values were 0.72 × 108 and 1.75 × 108 spores per gram of diet for 1st and 2nd instar larvae, respectively, and 3.84 × 108 spores per gram for adults. A significant increase in larval development time and reductions in pupal weight and emergence rate were observed when larvae were fed on diets containing sublethal concentrations of spores. Adults surviving after feeding on a sublethal diet showed a significant reduction in fecundity and longevity. Similarly, adults from treated larvae exhibited a lower fecundity, although their longevity was not influenced. A relationship between pupal weight and adult fecundity was found. Pupal stage duration and egg eclosion were not significantly affected.

Persistence and Effectiveness of Pyrethroids in Plastic Strips Against Varroa jacobsoni (Acari: Varroidae) and Mite Resistance in a Mediterranean Area

Abstract An apiary trial was conducted in 1997 in Sardinia, Italy, to verify the effectiveness of fluvalinate in polyvinyl chloride strips and flumethrin in polyethylene strips against Varroa jacobsoni Oudemans. Two indices to evaluate the efficacy of the treatments were adopted: percentage change in mite infestation of worker-sealed brood cells considering only treated hives and percentage change in mite mortality, and the natural variation in mite populations recorded in control hives during the trial. All acaricide treatments reduced the level of mite infestation of both sealed brood and adult bees. However, their effectiveness was slightly reduced in comparison to previous studies because of mite resistance phenomena. Portions of polyethylene strips of flumethrin from treated hives were sampled weekly to determine acaricide persistence using gas chromatography. After 4 wk, a slight reduction (≈9%) of the active ingredient content was observed. A laboratory bioassay also was performed to establish the resistance of adult female mites to fluvalinate. Mites were sampled from the experimental apiary and from various Sardinian apiaries which had primarily been subjected to fluvalinate applications in plastic strips or wood inserts for years. Mite resistance varied from 0 to 96%, depending on the acaricide management adopted. The lowest resistance level occurred in an apiary where pyrethroids had never been used, whereas the highest level occurred in an apiary with intensive use of fluvalinate in wood inserts.

Combination of beehive matrices analysis and ant biodiversity to study heavy metal pollution impact in a post-mining area (Sardinia, Italy)

Mining activities represent a major source of environment contamination. The aim of this study was to evaluate the use of bees and ants as bioindicators to detect the heavy metal impact in post-mining areas. A biomonitoring programme involving a combination of honeybee hive matrices analysis and ant biodiversity survey was conducted over a 3-year period. The experimental design involved three monitoring stations where repeated sampling activities focused on chemical detection of cadmium (Cd), chrome (Cr) and lead (Pb) from different matrices, both from hosted beehives (foraging bees, honey and pollen) and from the surrounding environment (stream water and soil). At the same time, ant biodiversity (number and abundance of species) was determined through a monitoring programme based on the use of pitfall traps placed in different habitats inside each mining site. The heavy metal content detected in stream water from the control station was always below the analytical limit of quantification. In the case of soil, the content of Cd and Pb from the control was lower than that of mining sites. The mean heavy metal concentrations in beehive matrices from mining sites were mainly higher than the control, and as a result of regression and discriminant analysis, forager bee sampling was an efficient environmental pollution bioindicator. Ant collection and identification highlighted a wide species variety with differences among habitats mostly associated with vegetation features. A lower variability was observed in the polluted landfill characterised by lack of vegetation. Combined biomonitoring with forager bees and ants represents a reliable tool for heavy metal environmental impact studies.

Observations on house fly larvae midgut ultrastructure after Brevibacillus laterosporus ingestion.

The pathological and histopathological course caused by Brevibacillus laterosporus on house fly larvae has been investigated conducting observations on insect behavior and midgut ultrastructure. After dissection and fixation, gut tissues were analyzed under transmission electron microscopy (TEM) in order to compare in vivo-treated and non-treated (control) fly specimens. Treated larvae showed extensively reduced feeding and growth rate, then became sluggish and died within 72 h. A progressive midgut epithelium deterioration was observed in treated larvae, compared to the control. Ultrastructural changes consisted of microvilli disruption, cytoplasm vacuolization and general disorganization, endoplasmic reticulum deformation, mitochondria alteration. Deterioration became progressively more dramatic until the infected cells released their content into the gut lumen. Disruption was associated also with midgut muscular sheath and connective tissue. These ultrastructural changes are similar to those widely described for other entomopathogenic bacteria, such as Bacillus thuringiensis, against different insect species. The rapid disruption of cellular fine structure supports a hypothesis based on an interaction of toxins with the epithelial cell membranes reminiscent of the specific B. thuringiensis δ-endotoxins mechanism of action on other insect targets.