Contamination of Honeybee (Apis mellifera L.) Royal Jelly by Pesticides and Sample Preparation Methods for Its Determination: A Critical Appraisal.

Pesticides can easily enter the food chain, harming bee populations and ecosystems. Exposure of beehive products to various contaminants has been identified as one of the factors contributing to the decline in bee populations, and multiple food alerts have been reported. Despite this fact, royal jelly, a valuable bee product with nutritional and functional properties, has received less attention in this context. Pesticide residues of different chemical class can contaminate royal jelly when foraging bees collect pollen or nectar from pesticide-treated flowers, or in some cases, due to its frequent and inappropriate use in the treatment of mites in beehives. To monitor this issue and also make it more reliable, it is crucial to develop effective sample preparation methods for extracting pesticides from royal jelly for subsequent analysis. In this context, this review provides information about sample preparation methods (solid-phase extraction, solvent extraction, and QuEChERS-quick, easy, cheap, effective, rugged and safe) and analytical methods that have been validated or improved to extract and analyze pesticides, respectively, in royal jelly samples of different origins. Finally, future perspectives are discussed. With this background, we aim to provide data that can guide future research related to this topic.

Monitoring changes in the volatile fraction of roasted nuts and seeds by using comprehensive two-dimensional gas chromatography and matrix templates.

Static headspace coupled with comprehensive two-dimensional gas chromatography and a flame ionization detector (HS-GC × GC-FID), has been applied to monitor changes in the volatile fraction of commercial edible nuts and seeds (peanuts, almonds, hazelnuts, and sunflower seeds). Effects of the roasting conditions (time, 5-40 min; temperature, 150-170 °C), which were employed under different combinations by using a ventilated oven, on target volatile fraction were examined to identify potential differences in relation to the roasting treatment of raw samples. In addition, reference templates were created, from the HS-GC × GC-FID method, for each of the four food matrices analyzed, and they were applied to characterize the samples according to the presence or absence of volatile compounds. Finally, these templates were successfully employed to make a quick distinction between different roasting conditions.•HS-GC × GC-FID was applied to study the volatile profile of edible nuts and seeds.•Reference templates (GC × GC-FID) were created for each of the four food matrices.•Rapid discrimination between raw and roasted samples was achieved.

Development and Validation of a Gas Chromatography-Mass Spectrometry Method for Determining Acaricides in Bee Pollen.

Pesticides can be found in beehives for several reasons, including contamination from surrounding crops or for their use by beekeepers, which poses a risk to bee ecosystems and consumers. Therefore, efficient and sensitive methods are needed for determining pesticide residues in bee products. In this study, a new analytical method has been developed and validated to determine seven acaricides (atrazine, chlorpyrifos, chlorfenvinphos, α-endosulfan, bromopropylate, coumaphos, and τ-fluvalinate) in bee pollen using gas chromatography coupled to mass spectrometry. After an optimization study, the best sample treatment was obtained when using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method employing an ethyl acetate and cyclohexane as the extractant mixture, and a mixture of salts for the clean-up step. A chromatographic analysis (<21 min) was performed in an Agilent DB-5MS column, and it was operated under programmed temperature conditions. The method was fully validated in terms of selectivity, limits of detection (0.2-3.1 µg kg-1) and quantification (0.6-9.7 µg kg-1), linearity, matrix effect (<20% in all cases), trueness (recoveries between 80% and 108%), and precision. Finally, the proposed method was applied to analyze commercial bee pollen samples, and some of the target pesticides (chlorfenvinphos, α-endosulfan, coumaphos, and τ-fluvalinate) were detected.

Determination of acaricides in honeys from different botanical origins by gas chromatography-mass spectrometry.

An analytical method has been proposed and validated to determine seven acaricides (atrazine, chlorpyrifos, chlorfenvinphos, α-endosulfan, bromopropylate, coumaphos, and τ-fluvalinate) in honeys from different botanical origins (multifloral, heather and rosemary) by means of gas chromatography-mass spectrometry. An efficient and simple sample treatment was proposed that involved a solvent extraction with an ethyl acetate and cyclohexane (50:50, v/v) mixture. Chromatographic analysis (<25 min) was performed in a DB-5MS column under programmed temperature conditions. The method was validated in terms of selectivity, limits of detection (0.2-2.0 µg kg-1) and quantification (0.5-7.6 µg kg-1), linearity (limit of quantification-700 (heather) or 800 (multifloral and rosemary) µg kg-1), matrix effect (<20 % in most cases), trueness (recoveries between 81 % and 108 %), and precision (relative standard deviation < 15 %). Finally, of the seven acaricides investigated in several honey samples only τ-fluvalinate residues (

Chiral and achiral separation of ten flavanones using supercritical fluid chromatography. Application to bee pollen analysis.

The separation of ten flavanones (flavanone, 2'-hydroxyflavanone, 4'-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, naringenin, naringin, hesperetin, pinostrobin, and taxifolin) using supercritical fluid chromatography and considering achiral and chiral approaches has been studied in this work. For this purpose, different stationary phases and organic modifiers have been checked. Considering the achiral separation, the best results were obtained with the Lichrospher 100 Diol column at 35 °C, 3 mL/min, 150 bar and a gradient of 2-propanol from 5% to 50%. The baseline separation of the ten compounds was achieved in 18 min. Using the chiral column Chiralpak AD, the separation of the ten pairs of enantiomers was obtained in 32 min. In this case, the chromatographic conditions were 30 °C, 3 mL/min, 150 bar and the organic modifier was a mixture ethanol/methanol (80:20) containing 0.1% of trifluoroacetic acid applied in an elution gradient from 15% to 50%. The applicability of the proposed chiral method was assessed by analysing bee pollen samples and 2S-pinostrobin was determined in some of them.

Differentiation of bee pollen samples according to the betaines and other quaternary ammonium related compounds content by using a canonical discriminant analysis.

In the last years, an increase has been observed in the adulteration of bee pollen. Consequently, different tools are required to authenticate the origin of this product, such as a study of the profile and composition of a specific family of compounds. The present study investigates the potential of betaines and related compounds as markers of the apiary of origin and harvest period of 71 bee pollen samples. These were collected from four apiaries (Pistacho, Tío Natalio, Monte and Fuentelahiguera), located in the same geographical area (Guadalajara, Spain) and sampled during three consecutive harvest periods in the same year (April-May, June, July-August). They were analyzed by means of a previously developed methodology, which involved solvent extraction, hydrophilic interaction liquid chromatography coupled to mass spectrometry, and a statistical analysis of the data (canonical discriminant analysis). Variable amounts of betaines and related compounds were found in the samples, with four of these being identified in all of them (betonicine, betaine, trigonelline and choline); betonicine was the predominant compound in a concentration range of 264 to 52384 mg/kg. It was possible to statistically assign over 50 % of the samples to the corresponding apiary of origin, the best results being obtained for the Tío Natalio apiary (75 %); this classification was even better in the case of the harvest period, as more than 75 % of the samples were correctly assigned, and in two periods (April-May and June) a 90 % rate was obtained.

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.

Polymeric stationary phases based on poly(butylene terephthalate) and poly(4-vinylpirydine) in the analysis of polyphenols using supercritical fluid chromatography. Application to bee pollen.

Two new polymer-based stationary phases; DCpak PBT (poly(butylene terephthalate)) and DCpak P4VP (poly(4-vinylpirydine)) were evaluated for the analysis of polyphenols using supercritical fluid chromatography (SFC). The compounds studied included phenolic acids and flavonoids. The different variables that influence the chromatographic separation, such as type and percentage of organic modifier, additive, pressure and temperature were examined. Using the DCpak P4VP column the retention was exceptionally high, obtaining better results with the DCpak PBT column. The separation of nine polyphenols was achieved using a gradient of modifier (methanol with 0.1% trifluoroacetic acid) from 5 to 50%, a pressure of 150 bar, a temperature of 35 °C and a flow-rate of 2 mL/min. The use of additives was necessary in order to obtain good peak shapes and efficiencies, achieving the best results with trifluoroacetic acid. LODs and LOQs values were lower than 5 µg/mL in all the cases; meanwhile, the %RSD values for method repeatability and inter-day reproducibility were lower than 3% and 10% respectively. Finally, the proposed method was successfully applied to the analysis of polyphenols in commercial bee pollen; four compounds, namely cinnamic acid, p-coumaric acid, catechin and quercetin were identified and quantified.