María J. Nozal

Honeybee colony collapse due to Nosema ceranae in professional apiaries.

Honeybee colony collapse is a sanitary and ecological worldwide problem. The features of this syndrome are an unexplained disappearance of adult bees, a lack of brood attention, reduced colony strength, and heavy winter mortality without any previous evident pathological disturbances. To date there has not been a consensus about its origins. This report describes the clinical features of two professional bee-keepers affecting by this syndrome. Anamnesis, clinical examination and analyses support that the depopulation in both cases was due to the infection by Nosema ceranae (Microsporidia), an emerging pathogen of Apis mellifera. No other significant pathogens or pesticides (neonicotinoids) were detected and the bees had not been foraging in corn or sunflower crops. The treatment with fumagillin avoided the loss of surviving weak colonies. This is the first case report of honeybee colony collapse due to N. ceranae in professional apiaries in field conditions reported worldwide.

A preliminary study of the epidemiological factors related to honey bee colony loss in Spain

In recent years, a worldwide decline in the Apis mellifera populations has been detected in many regions, including Spain. This decline is thought to be related to the effects of pathogens or pesticides, although to what extent these factors are implicated is still not clear. In this study, we estimated the prevalence of honey bee colony depopulation symptoms in a random selected sample (n = 61) and we explored the implication of different pathogens, pesticides and the flora visited in the area under study. The prevalence of colony depopulation symptoms in the professional apiaries studied was 67.2% [95% confidence interval (CI) = 54.6-79.8; P < 0.0001]. The most prevalent pathogen found in the worker honey bee samples was Nosema ceranae[65.6%; 95% CI = 52.8-78.3; P < 0.0001], followed by Varroa destructor[32.7%; 95% CI = 20.2-45.4; P < 0.0001] and 97.5% of the colonies infected by N. ceranae were unhealthy (depopulated). Co-infection by V. destructor and N. ceranae was evident in 22.9% (95% CI = 11.6-34.3; P < 0.0001) of the samples and only in unhealthy colonies. Of the 40 pesticides studied, only nine were detected in 49% of the stored pollen samples analysed. Fipronil was detected in only three of 61 stored pollen samples and imidacloprid was not detected in any. Acaricides like fluvalinate, and chlorfenvinphos used to control Varroa mite were the most predominant residues in the stored pollen, probably as a result of their application in homemade formulae. None of the pesticides identified were statistically associated to colony depopulated. This preliminary study of epidemiological factors suggests that N. ceranae is a key factor in the colony losses detected over recent years in Spain. However, more detailed studies that permit subgroup analyses will be necessary to contrast these findings.

High-performance liquid chromatographic determination of methyl anthranilate, hydroxymethylfurfural and related compounds in honey☆

A high-performance liquid chromatographic method for determining 5-hydroxymethyl-2-furaldehyde (hydroxymethylfurfural), 2-furaldehyde (furfural), furan-2-carboxylic acid (2-furoic acid), furan-3-carboxylic acid (3-furoic acid), furan-3-carboxaldehyde (3-furaldehyde) and 2-aminobenzoic acid methyl ester (methyl anthranilate) in honey and honeydew samples is described. To prevent matrix interference and to isolate the compounds, a clean-up step which implies a solid-phase extraction on polymeric cartridges and an elution with 0.5 ml methanol is recommended. The compounds are separated on a reversed-phase column with a gradient of (A) 1% aqueous acetic acid-acetonitrile (97:3, v/v) and (B) acetonitrile-water (50:50, v/v), with UV detection at 250 nm. The method is applied to the analysis of samples from different botanical origin.

Determination of seven neonicotinoid insecticides in beeswax by liquid chromatography coupled to electrospray-mass spectrometry using a fused-core column.

A new method has been developed to measure seven neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) in beeswax using liquid chromatography (LC) coupled to electrospray ionization mass spectrometry (ESI-MS) detection. Beeswax was melted and diluted in an n-hexane/isopropanol (8:2, v/v) mixture. After this, liquid extraction with water was performed followed by a clean-up on diatomaceous material based cartridges. The compounds were eluted with acetone, and the resulting solution was evaporated until dry and reconstituted with a mixture of water and acetonitrile 50:50 (v/v). The separation of all compounds was achieved in less than 15 min using a C18 reverse-phase fused-core column (Kinetex C18, 150 mm × 4.6 mm i.d.) and a mobile phase composed of a mixture of 0.1% formic acid in water and acetonitrile in gradient elution mode at 0.5 mL/min. This method was fully validated in terms of selectivity, linearity, precision and recovery. Low limits of detection and quantification could be achieved for all analytes ranging from 0.4 to 2.3 μg/kg, and from 1.5 to 7.0 μg/kg, respectively. Finally, the proposed method was applied to an analysis of neonicotinoid residues in beeswax samples from apiaries located close to fruit orchards.

Development and validation of an LC assay for sumatriptan succinate residues on surfaces in the manufacture of pharmaceuticals

A high performance liquid chromatographic (HPLC) method for the assay of sumatriptan succinate residues in swabs collected from manufacturing equipment surfaces was developed and validated in order to control a cleaning procedure. The swabbing procedure using two cotton swabs moistened with water was validated applying a wipe-test and a HPLC method developed to determine low quantities of the drug. The HPLC method involves a C18 column at 25 degrees C, a mixture of ammonium phosphate monobasic (0.05 M)-acetonitrile (84:16, v/v) as a mobile phase and UV detection at 228 nm. Using the proposed method, the average recoveries obtained are of 88.5% for vinyl, 94.2% for glass and 95.2% for stainless steel plates with RSD of 5.5 (n=36), 2.3 (n=36), 2.2% (n=36), respectively. The method was successfully applied to the assay of real swab samples collected from the equipment surfaces.

Presence of mitomycin-C in the anterior chamber after photorefractive keratectomy

PURPOSE: To assess the presence of mitomycin‐C (MMC) in hen aqueous humor after photorefractive keratectomy (PRK). SETTING: Instituto Universitario de Oftalmobiología Aplicada, Faculty of Medicine, University of Valladolid, and Department of Analytical Chemistry, Faculty of Sciences, University of Valladolid, Valladolid, Spain. METHODS: Mitomycin‐C 0.02% was applied topically for 2 minutes to a right hens eye after PRK (Group A) and to the left eye with intact epithelium (Group B). At different time points (10, 30, 60, 360, and 720 minutes), aqueous humor was extracted and high‐performance liquid chromatography was performed to detect and quantify MMC levels. RESULTS: The mean maximum drug concentration of MMC measured in the aqueous humor was 187.250 μg/L ± 4.349 (SD) in Group A and 93.000 ± 4.899 μg/L in Group B, both detected 10 minutes after topical application. Statistically significant differences were found between Groups A and B at 10, 30, and 60 minutes, with decreasing MMC levels in both groups but a higher concentration in Group A. After 360 minutes, MMC levels were undetectable in Group B and after 720 minutes in Group A. CONCLUSIONS: Mitomycin‐C was detectable in the aqueous humor of the hen eye after topical application in PRK‐treated eyes and in eyes with intact epithelium. The presence of MMC is of concern as it may lead to ocular toxicity in the long term.

Determination of glutathione, cysteine and N-acetylcysteine in rabbit eye tissues using high-performance liquid chromatography and post-column derivatization with 5,5′-dithiobis(2-nitrobenzoic acid)

A high-performance liquid chromatographic method to determine glutathione, cysteine and N-acetylcysteine in rabbit retina, vitreous and lens has been developed. The thiols are separated using a 25 x 0.46-cm octadecylsilane column with 0.5 M phosphate buffer, pH 3, as mobile phase. The detection, at 412 nm, involves a post-column derivatization with 5,5-dithiobis(2-nitrobenzoic acid) in presence of cationic micelles of hexadecyltrimethylammonium bromide that enhances the sensitivity. The detection limits are 0.21, 0.92 and 0.61 mumol/g wet sample for glutathione, cysteine and N-acetylcysteine, respectively.

Extraction, chemical characterization and biological activity determination of broccoli health promoting compounds.

Broccoli (Brassica oleracea L. var. Italica) contains substantial amount of health-promoting compounds such as vitamins, glucosinolates, phenolic compounds, and dietary essential minerals; thus, it benefits health beyond providing just basic nutrition, and consumption of broccoli has been increasing over the years. This review gives an overview on the extraction and separation techniques, as well as the biological activity of some of the above mentioned compounds which have been published in the period January 2008 to January 2013. The work has been distributed according to the different families of health promoting compounds discussing the extraction procedures and the analytical techniques employed for their characterization. Finally, information about the different biological activities of these compounds has been also provided.

A new and simple method to determine trace levels of sulfonamides in honey by high performance liquid chromatography with fluorescence detection.

A novel method for the simultaneous analysis at trace level of sulfonamides (sulfaguanidine, sulfanilamide, sulfacetamide, sulfathiazole, sulfapyridine, sulfachloropyridazine, sulfamerazine, sulfameter, sulfamethazine, sulfadoxine, sulfadiazine, sulfamonomethoxine, sulfadimethoxine) in honey is described. Methanol has been used in the sample treatment step to avoid the emulsion formation and to break the N-glycosidic bond between sugars and sulfonamides. The determination is carried out by liquid chromatography in gradient elution mode, with fluorescence detection after the on-line pre-column derivatization with fluorescamine. The influence of parameters such as the mobile phase composition, column temperature, pH or injection volume, on the separation has been taken into account and the derivatization step has also been optimized. Recoveries of the compounds on spiked honey samples ranged from 56% for sulfadoxine to 96% for sulfacetamide, with relative standard deviations below 10%. The quantitation limits are between 4 and 15 ng g(-1).

Profile and relative concentrations of fatty acids in corn and soybean seeds from transgenic and isogenic crops.

In this work 44 fatty acids, which were analyzed as methyl esters by GC/MS in scan mode, have been determined in genetically modified corn and soybean seeds. Their relative concentrations have been compared with those of isogenic lines grown in the same conditions. Studied compounds comprised saturated and unsaturated fatty acids, including cis/trans isomers and minor fatty acids. A classical soxhlet extraction and an accelerated solvent extraction have been assayed to extract the fatty compounds from seeds and the GC separation has been carried out on a biscyanopropylpolysiloxane chromatographic column. Soxhlet extraction was selected as the most convenient and applied to compare the samples. Specific compounds, which could denote the origin of the crop have not been observed, but for some sample pairs, significant differences have been found in relation to the percentage of certain acids; the highest differences for major acids were 4.1% in corn and 4.8% in soybean. The concentrations of long chain acids such as 24:0, 26:0 and 28:0 were higher in some isogenic lines whereas the concentrations of short chain acids such as 6:0, 8:0, 9:0, 10:0 and 12:0 were higher in their transgenic counterparts.