Simultaneous determination of carvacrol and thymol in bee pollen by using a simple and efficient solvent extraction method and gas chromatography-mass spectrometry.

A novel method is proposed to determine residues of carvacrol and thymol in bee pollen by means of gas chromatography coupled to mass spectrometry. This is an efficient and simple sample treatment (with average analyte recoveries between 90% and 104%) involving solvent extraction with hexane followed by evaporation. There is no need for any additional clean-up step, as the matrix did not affect determination of mass spectrometry for either compound. The chromatographic conditions are also optimized: a ZB-WAX column is employed, helium is the carrier gas at a flow rate of 1.1 mL/min, and a temperature program is included, allowing baseline separation of both compounds in less than 21 min. The method is fully validated in terms of selectivity, limits of detection and quantification, matrix effect, linearity, precision and trueness. Results show that not only is it selective, but that it also displays a wide linearity range (limit of quantification-1000 µg/kg), good precision (relative standard deviation values lower than 8%) and sensitivity (limits of detection and quantification lower than 15 µg/kg). Finally, several bee pollen samples are analysed, and thymol and carvacrol residues are found at low concentrations (limit of quantification-57 µg/kg) in some cases.

Extraction and determination of bioactive compounds from bee pollen.

Since ancient times bee pollen has been considered a good source of bioactive substances and energy. Taking into account the current demand for healthy and natural foods, it is not surprising that bee pollen has been attracting commercial interest in recent years, making it one of the most widely consumed food supplements. It has been extensively reported that bee pollen contains several health-promoting compounds, such as proteins, amino acids, lipids, phenolic compounds, vitamins or minerals. Thus, this study aims to give an overview of the extraction and determination techniques of several of the above-mentioned compounds which have been published in the last few years (2011-2017). The design of the study is in accordance with the different families of bioactive compounds, and the extraction procedures together with the analytical techniques employed and their determination are discussed. A list of some of the most relevant applications is provided for each category, including a brief summary of the experimental conditions. The references included will provide the reader with a comprehensive overview of and insight into the analysis of bioactive compounds from bee pollen.

Trace analysis of sulforaphane in bee pollen and royal jelly by liquid chromatography-tandem mass spectrometry.

In this study, we investigate for the first time the presence of sulforaphane (SFN) residues in two of the most currently consumed food/dietary supplements, royal jelly and bee pollen. Chromatography-tandem mass spectrometry (LC-MS/MS) was the method employed, the mass spectrometer consisting of an ion-trap mass analyzer used with electrospray ionization (ESI) in positive ion mode. An efficient sample treatment involving a solvent extraction with methanol, centrifugation, and concentration in a rotary evaporator was proposed. In all cases average analyte recoveries were between 92 and 106%. Chromatographic analysis (16min) was performed on a core-shell technology based column (Kinetex C18, 150×4.6mm, 2.6µm, 100Å). The mobile phase consisted of 0.02M ammonium formate in water and acetonitrile, with a flow rate of 0.5mL/min in gradient elution mode. The fully validated method was selective, linear from 8 to 1000µg/kg (bee pollen), or from 10 to 1250µg/kg (royal jelly), precise and accurate; relative standard deviation (% RSD) and relative error (% RE) values were below 10%. Low limits of detection (LOD) and quantification (LOQ) were obtained, namely, 3µg/kg (LOD) and 8 (bee pollen) and 10 (royal jelly) µg/kg (LOQ). The method was applied for SFN analysis in several royal jelly and bee pollen samples. SFN was detected at trace levels in some bee pollen samples (<23µg/kg) examined, whilst SFN went undetected in the royal jelly samples analyzed.

Residues of neonicotinoids and their metabolites in honey and pollen from sunflower and maize seed dressing crops.

A study was carried out to evaluate the possible presence of thiamethoxam, clothianidin and imidacloprid, as well as the metabolic breakdown products of these three neonicotinoids in pollen and honey obtained from brood chamber combs of honeybee colonies located next to sunflower and maize crops from coated seeds. Samples were analyzed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry detector, in combination with accurate mass tools such as diagnostic ions by exact mass, chlorine mass filters, and MS/MS experiments. The presence of thiamethoxam and clothianidin was confirmed in some of the pollen samples analyzed. Moreover, different metabolites of neonicotinoids were tentatively detected in the pollen and honey samples collected. The results suggested that four metabolites were found in the honey samples, while for pollen samples eleven metabolites were identified; among these, five were considered for the first time as metabolic breakdown products in sunflower and maize plants.

Development and validation of a liquid chromatography-tandem mass spectrometry method to determine intact glucosinolates in bee pollen.

A new method was developed to determine twelve intact-glucosinolates (GLSs) (glucoiberin, GIB; glucoraphanin, GRA; glucoerucin GER; gluconapin, GNA; glucotropaeolin, GTL; glucobrassicin, GBC; gluconasturtiin, NAS; glucoalyssin, ALY; 4-hydroxyglucobrassicin, 4OH; 4-methoxyglucobrassicin, 4ME; neoglucobrassicin, NEO; sinigrin, SIN) in bee pollen, by means of liquid chromatography tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). An efficient extraction procedure was proposed (average analyte recoveries were between 85% and 96%); this involved a solid-liquid extraction (SLE) with heated water, followed by a solid phase extraction (SPE) with a weak anion exchange (NH2) sorbent. Chromatography was performed on a Gemini(®) C18 analytical column with a mobile phase of formic acid in water (0.5%,v/v) and formic acid in acetonitrile (0.5%,v/v), in gradient elution mode at 1mL/min, resulted in baseline-separated peaks and a run time of 30min. The method was fully validated in terms of selectivity, limits of detection (LOD) and quantification (LOQ), linearity, carry-over effect, reinjection reproducibility, precision and accuracy. A good selectivity, low LODs and LOQs, ranging from 1 to 16µg/kg, wide linear ranges from LOQ to 1000µg/kg, and satisfactory reinjection reproducibility, precision and accuracy with relative standard deviation and relative error values lower than or equal to 9%; meanwhile, results indicates a negligible carry-over effect. The proposed method was applied to analyze intact-GLSs in bee pollen. Nine of the GLSs studied were identified in certain samples analyzed over a wide concentration range (LOQ-2226µg/kg), and significant differences in GLS content were observed among the samples.

Holistic screening of collapsing honey bee colonies in Spain: a case study.

BACKGROUND: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse. RESULTS: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly. CONCLUSION: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified.

Optimized extraction, separation and quantification of twelve intact glucosinolates in broccoli leaves.

A new method has been developed and validated to determine twelve intact glucosinolates (glucoiberin, GIB; glucoraphanin, GRA; glucoerucin GER; gluconapin, GNA; glucotropaeolin, GTL; glucobrassicin, GBC; gluconasturtiin, GST; glucoalyssin, ALY; 4-hydroxyglucobrassicin, 4-OH; 4-metoxyglucobrassicin, 4ME; neoglucobrassicin, NEO; sinigrin, SIN) in broccoli leaves using liquid chromatography (LC) coupled to diode array (DAD) and electrospray ionization mass spectrometry (ESI-MS) detection. An extraction procedure has also been proposed and optimized by means of statistical analysis (the Box-Behnken design and analysis of variance); this is based on the deactivation of myrosinase using a microwave and heated water. Low limits of detection and quantification were obtained, ranging from 10 to 72 µg/g with DAD and 0.01 to 0.23 µg/g with ESI-MS, and the resulting recovery values ranged from 87% to 106% in all cases. Finally, glucosinolates were analyzed in broccoli leaf samples from six different cultivars (Ramoso calabrese Parthenon, Marathon, Nubia, Naxos and Viola).

Determination of spinosad at trace levels in bee pollen and beeswax with solid-liquid extraction and LC-ESI-MS.

This paper reports the use of a new LC method with a fused-core analytical column coupled to ESI-MS to determine residues of the biopesticide spinosad in bee pollen and beeswax. The method analyzes the active ingredients, spinosyns A and D, with a simple and efficient sample treatment (recovery between 90 and 105%) consisting of a solid-liquid extraction with acetone (bee pollen) or acetonitrile (beeswax). The method was validated in terms of selectivity, LOD, LOQ, linearity, and precision. The LOD and LOQ values ranged between 0.1-0.2 and 0.4-0.7 µg/kg, respectively. Moreover, the precision obtained within the linear concentration range (LOQ 500 µg/kg) was satisfactory (RSD lower than 5%). Finally, the proposed method was applied to analyze bee pollen and beeswax samples collected from apiaries located close to fruit orchards in two Spanish regions.

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.

Development and application of a liquid chromatography-mass spectrometry method to evaluate the glyphosate and aminomethylphosphonic acid dissipation in maize plants after foliar treatment.

A simple and fast method has been developed and validated to measure glyphosate (GLYP) and aminomethylphosphonic acid (AMPA), which were previously derivatized with 9-fluorenylmethylchloroformate (FMOC-Cl), in maize plants using liquid chromatography (LC) coupled to fluorescence (FLD) and electrospray ionization mass spectrometry (ESI-MS) detection. The method has shown to be consistent, reliable, precise, and efficient. Moreover, the limits of detection (LOD) and quantification (LOQ) reached with the proposed method for GLYP and AMPA are lower than the established maximum residue levels (MRLs). The validated method was applied to quantify GLYP and AMPA in genetically modified (GM) maize foliar treated with the herbicide. It has been found that the GLYP dissipation was mainly due to the progressive dilution effect after herbicide treatment. Finally, it was also observed that the GLYP residue dissipation trend in maize shoot (leaves and stem) tissue determined by LC-ESI-MS matched that determined by liquid scintillation.

An exposure study to assess the potential impact of fipronil in treated sunflower seeds on honey bee colony losses in Spain.

BACKGROUND: There is great concern about the high losses and strong depopulation of honey bee colonies in some areas of Spain. Some beekeepers have suggested that sunflower seeds treated with the insecticide fipronil could be an important factor in causing those losses. Therefore, an in-depth field study has been carried out in two regions of Spain where sunflower production is intense (Cuenca and Andalucía) and where, for some crops and varieties, fipronil has been used as seed insecticide. RESULTS: Samples of adult bees and pollen were analysed for bee pathogens and pesticide residues respectively. Neither fipronil residues nor its metabolites were detected in any of the samples analysed, indicating that short-term or chronic exposure of bees to fipronil and/or its metabolites can be ruled out in the apiaries surveyed. Varroa destructor and Nosema ceranae were found to be very prevalent. CONCLUSION: The combination of the two pathogens could augment the risk of colony death in infected colonies, without fipronil residues exerting a significant effect in the given field conditions. Indeed, in this study the losses observed in apiaries located close to sunflower crops were similar to those in apiaries situated in forested areas with wild vegetation.