Intact Glucosinolates Research Articles

The role of water in the impact of high pressure on the myrosinase activity and glucosinolate content in seedlings from Brussels sprouts

Abstract In this study, it was shown that the amount of available water was found to influence the high pressure processing (HPP) effect on both myrosinase activity and total glucosinolate concentration in Brussels sprouts seedlings. Brussels sprouts seedlings with different water content (wc = 4.8–89.4%) and water activity (aw = 0.17–0.97) were pressurized at selected pressures between 200 and 800 MPa (5 °C and 3 min), thereby affecting pressure-induced enzyme denaturation, molecular diffusion, and cell permeability differently. The myrosinase activity and intact glucosinolate content in the dry seedlings (wc Industrial relevance High pressure processing (HPP) is increasingly applied in the food industry. The treatment is acknowledged for the ability to give products longer shelf life concomitant with a high nutritional quality and fresh appearance. Upon HPP of cruciferous plants it is important to have a special focus on the glucosinolate-myrosinase system, since sub-optimal pre-treatment and/or processing parameters can affect the food quality negatively. The present study provides valuable results regarding the significance of water content and activity on the sensitivity of the myrosinase-glucosinolate system in Brussels sprouts seedlings towards HPP. Thus, providing a tool for designing different types of HPP products with respect to levels of active myrosinase and intact glucosinolates by adjustment of water content, water activity and HPP level.

Antimicrobial and Cytotoxic Activities of Lepidium latifolium L. Hydrodistillate, Extract and Its Major Sulfur Volatile Allyl Isothiocyanate

The cultivated Lepidium latifolium L. was investigated to decipher its glucosinolate profile, antimicrobial, and cytotoxic activities. HPLC/ESI-MS analyses of the intact glucosinolates and GC/MS analysis of their hydrolysis products showed the presence of sinigrin (1), glucocochlearin (2), glucotropaeolin (3), and 4-methoxyglucobrassicin (4). Hydrodistillate, extract, and allyl isothiocyanate, the main volatile resulting from sinigrin degradation, showed antimicrobial activity against all eleven tested pathogenic and food spoilage bacteria and fungi, with highest effect observed against Candida albicans with MIC50 8 and 16 μg/mL. Hydrodistillate and extract showed the best cytotoxic activity on bladder cancer UM-UC-3 cell line during an incubation time of 24 h (IC50 192.9 and 133.8 μg/mL, respectively), while the best effect on glioblastoma LN229 cell line was observed after 48 h (IC50 110.8 and 30.9 μg/mL, respectively). Pure allyl isothiocyanate displayed a similar trend in cytotoxic effect on both cell lines (IC50 23.3 and 36.5 μg/mL after 24 h and 48 h, respectively).

Determination of glucosinolates in broccoli-based dietary supplements by cyclodextrin-mediated capillary zone electrophoresis

A new cyclodextrin-mediated capillary zone electrophoresis method for the separation and quantification of intact glucosinolates (GSL) has been developed. β-Cyclodextrin concentration and temperature were shown to be key parameters for selectivity. Controlled variation of both parameters provided a set of methods for the analysis of GSL-containing plants with customized selectivities. As a proof of concept, parameters were adapted for the analysis of broccoli-based dietary supplements (BBDS) obtained from different sources. All main GSL (glucoraphanin, -iberin, -erucin, and 4-hydroxyglucobrassicin) – which typically occur in broccoli plants at early development stages – were successfully analyzed using a 100 mM borate buffer with 1% β-cyclodextrin at 20 °C.Analysis of BBDS showed unexpected results. In most cases, supplements had been specified to contain seeds, sprouts or young broccoli plants. In many cases, results did not reflect the description on the label. Several samples apparently contained broccoli from older plant development stages, marked by higher amounts of indole-derived GSL and very low concentrations of aliphatic GSL. Considerable amounts of non-broccoli GSL were detected in two samples: sinalbin and a mixture of 3-butenyl- and 4-pentenylglucosinolate. No glucosinolates were detectable in two further samples. In total >50% of all investigated samples did not meet the specifications.

Improved Separation of Intact Glucosinolates in Bee Pollen by Using Ultra-High-Performance Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry

A method to determine 15 intact glucosinolates (epiprogoitrin; glucoalyssin; glucobrassicanapin; glucobrassicin; glucoerucin; glucoiberin; gluconapin; gluconasturtiin; glucoraphanin; glucotropaeolin; 4-hydroxyglucobrassicin; 4-methoxyglucobrassicin; neoglucobrassicin; progoitrin; sinigrin) in bee pollen has been proposed, by means of ultra-high liquid chromatography coupled to a quadrupole time-of-flight mass detector. Efficient sample treatment (with average analyte recoveries between 80 and 96%) involving a solid-liquid extraction with hot water, followed by solid-phase extraction with a weak anion exchange sorbent (NH2), was employed to analyze those intact glucosinolates in bee pollen. Chromatographic analysis (15 min) was performed on a fully porous silica–based column (Luna® Omega C18) with a mobile phase composed of a mixture of acetonitrile (0.1% formic acid) and water (0.1% formic acid) in gradient elution mode at a flow rate of 0.2 mL/min. Accordingly, the analytical performance of the proposed method was evaluated, and it was then applied to determine intact glucosinolates in commercial bee pollen samples from different Spanish regions. Ten intact glucosinolates were identified in certain samples over a wide concentration range.

Glucosinolates in Draba borealis DC. (Brassicaceae) in a taxonomic perspective

Draba borealis DC. is a perennial wild plant from the Arctic regions of Canada and USA. The seed and a whole plant of D. borealis were extracted and analysed by LC-MS for intact glucosinolates (GLs). Several identifications were confirmed by isolation. The identification of eleven compounds is reported in the seed and in the whole plant, including 3-methylsulfinylpropyl- (1), 4-methylsulfinylbutyl- (2), 5-methylsulfinylpentyl- (3), 3-methylsulfanylpropyl- (5), 6-methylsulfinylhexyl- (6), 5-methylsulfanylpentyl- (7), 6-methylsulfanylhexyl- (8), 4-methylsulfanylbutyl- (9), 8-methylsulfinyloctyl- (10), 9-methylsulfinylnonyl- (11), and 7-methylsulfanylheptyl- (12). Two additional GLs, 1-methylpropyl- (4) and 7-methylsulfinylheptyl- (13) GLs, were also proposed. This is the first report of occurrence of GLs 1–3, 5–9 and 12–13 from the genus Draba. Finally, the taxonomic perspective of the GL side chains in Draba genus was also discussed.

Chromatographic column evaluation for the untargeted profiling of glucosinolates in cauliflower by means of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry

The untargeted profiling is a promising approach for the characterization of secondary metabolites in biological matrices. Thanks to the recent rapid development of high-resolution mass spectrometry (HRMS) instrumentations, the number of applications by untargeted approaches for biological samples profiling has widely increased in the recent years. Despite the high potentialities of HRMS, however, a major issue in natural products analysis often arises in the upstream process of compounds separation. A separation technique is necessary to avoid phenomena such as signal suppression, and it is especially needed in the presence of isomeric metabolites, which are otherwise indistinguishable. Glucosinolates (GLSs), a group of secondary metabolites widely distributed among plants, resulted to be associated to the prevention of some serious diseases, such as cancer. This led to the development of several methods for the analysis of GLSs in vegetables tissues. The issue of GLSs chromatographic separation has been widely studied in the past because of the difficulty in the analysis of this highly polar and variable class of compounds. Several alternatives to reversed phase (RP) chromatography, sometimes not compatible with the coupling of liquid chromatography with mass spectrometry, have been tested for the analysis of intact GLSs. However, the availability of new stationary phases, in the last years, could allow the re-evaluation of RP chromatography for the analysis of intact GLSs.In this work, a thorough evaluation of four RP chromatographic columns for the analysis of GLSs in cauliflower (Brassica oleracea L. var. botrytis) extracts by an ultra-high performance liquid chromatographic system coupled via electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer is presented. The columns tested were the following: one column Luna Omega polar C18, one column Kinetex Biphenyl, one column Kinetex core-shell XB-C18, two columns Kinetex core-shell XB-C18. After a previous optimization of the extraction method, cauliflower extracts were analyzed testing four different mobile phases onto the four columns for a total of sixteen different chromatographic conditions. The chromatographic systems were evaluated based on the number of detected and tentatively identified GLSs. Luna Polar stationary phase resulted to be the most suitable for the analysis of GLSs compared to Kinetex XB and Kinetex Biphenyl columns stationary phase. However, two in series Kinetex XB columns increased the number of tentatively identified GLSs compared to one Kinetex XB, showing the importance of column length in the analysis of complex mixtures. The data obtained with the best chromatographic system were deeply analyzed by MS/MS investigation for the final identification. Fiflty-one GLSs were tentatively identified, 24 of which have never been identified in cauliflower. Finally the linearity of the analytes response over the analyzed range of concentration was checked, suggesting that the developed method is suitable for both qualitative and quantitative analysis of GLSs in phytochemical mixtures.

Solubility of a cruciferin‐rich protein product purified from rapeseed pressed cake (Brassica napus L.) by an aqueous processing method

SummaryPressed cake from rapeseed (Brassica napus L.) is a promising plant protein source not yet utilised for human consumption due to the presence of antinutrients such as glucosinolates. Protein solubility is a crucial parameter influencing the functionality and thereby the applicability of proteins as food ingredients. A novel cruciferin‐rich rapeseed protein product was produced by an aqueous processing method in pilot plant scale. Intact glucosinolates were conserved by this procedure and largely removed from the protein products. Protein solubility in this product was examined when dispersed in 50 mm phosphate buffer, pH 8.0 with varying NaCl concentration (0–500 mm). Unexpectedly, a salting‐out effect was observed of the globulin proteins, as 15.9 ± 0.6% protein was in solution at 500 mm added NaCl, whereas 21.5 ± 1.1% was solubilised without added NaCl; whether the observed effects originates from lipid and fibre constituents in the product remains to be resolved.

Metabolic profiling of glucosinolates and their hydrolysis products in a germplasm collection of Brassica rapa turnips

About 10% of the world's vegetable production is generated from Brassicaceae, wherein Brassica rapa is a dominating species. There is growing evidence that glucosinolates (GLSs), main plant secondary metabolites in Brassicales, play an important role in promoting human health. Natural genetic diversity of B. rapa can be explored for vegetable improvement. We analyzed leaves and tubers of 16 B. rapa turnips for their GLS composition by UHPLC-DAD and the corresponding hydrolysis products by GC–MS. Thirteen GLSs were identified, 8 aliphatic, 4 indolic and one aromatic. 3-Butenyl GLS was prevailing in both plant organs while in tubers 2-hydroxy-3-butenyl GLS and 2-phenylethyl GLS occurred in high amounts. A total of 24 GLS breakdown products were detected in tubers and 16 in leaves. Epithionitriles were the main hydrolysis products in both plant organs with 4,5-epithiopentanenitrile and 3-hydroxy-4,5-epithiopentanenitrile being the main compounds. When comparing leaves and tubers, an accumulation of GLSs and their breakdown products was observed in tubers compared to leaves. Our analysis achieved the comprehensive profiling of all GLS metabolites in a collection of B. rapa turnips, underlining the natural variation not only of intact GLS, but also of their breakdown products.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC).

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

Quantification and bioaccessibility of intact glucosinolates in broccoli ‘Parthenon’ and Savoy cabbage ‘Dama’

Brassica has demonstrated to exhibit health benefits due to their content in bioactive compounds, such as glucosinolates (GS) among others. The aim of this work was to identify and quantify the intact GS of two brassicas, broccoli ‘Parthenon’ and Savoy cabbage ‘Dama’. Additionally, the bioaccessibility of these compounds was evaluated by in vitro digestion procedure simulating gastric and small intestinal digestion. Glucobrassicin and glucobrassicanapin were the most abundant GS in both brassicas. The aromatic GS gluconasturtiin was only detected in broccoli. After in vitro gastrointestinal digestion, losses in GS content were found, 63% for broccoli and 69% for Savoy cabbage. The highest percentage of bioaccessibility was for the GS glucobrassicin in both brassicas (∼42%). In general, GS bioaccessibility was higher in broccoli ‘Parthenon’ than in Savoy cabbage ‘Dama’.

Rhizosecretion of stele-synthesized glucosinolates and their catabolites requires GTR-mediated import in Arabidopsis

Casparian strip-generated apoplastic barriers not only control the radial flow of both water and ions but may also constitute a hindrance for the rhizosecretion of stele-synthesized phytochemicals. Here, we establish root-synthesized glucosinolates (GLS) are in Arabidopsis as a model to study the transport routes of plant-derived metabolites from the site of synthesis to the rhizosphere. Analysing the expression of GLS synthetic genes in the root indicate that the stele is the major site for the synthesis of aliphatic GLS, whereas indole GLS can be synthesized in both the stele and the cortex. Sampling root exudates from the wild type and the double mutant of the GLS importers GTR1 and GTR2 show that GTR-mediated retention of stele-synthesized GLS is a prerequisite for the exudation of both intact GLS and their catabolites into the rhizosphere. The expression of the GTRs inside the stele, combined with the previous observation that GLS are exported from biosynthetic cells, suggest three possible routes of stele-synthesized aliphatic GLS after their synthesis: (i) GTR-dependent import to cells symplastically connected to the cortical cells and the rhizosphere; (ii) GTR-independent transport via the xylem to the shoot; and (iii) GTR-dependent import to GLS-degrading myrosin cells at the cortex. The study suggests a previously undiscovered role of the import process in the rhizosecretion of root-synthesized phytochemicals.

GLS-Finder: A Platform for Fast Profiling of Glucosinolates in Brassica Vegetables.

Mass spectrometry combined with related tandem techniques has become the most popular method for plant secondary metabolite characterization. We introduce a new strategy based on in-database searching, mass fragmentation behavior study, formula predicting for fast profiling of glucosinolates, a class of important compounds in brassica vegetables. A MATLAB script-based expert system computer program, "GLS-Finder", was developed. It is capable of qualitative and semi-quantitative analyses of glucosinolates in samples using data generated by ultrahigh-performance liquid chromatography-high-resolution accurate mass with multi-stage mass fragmentation (UHPLC-HRAM/MS(n)). A suite of bioinformatic tools was integrated into the "GLS-Finder" to perform raw data deconvolution, peak alignment, glucosinolate putative assignments, semi-quantitation, and unsupervised principal component analysis (PCA). GLS-Finder was successfully applied to identify intact glucosinolates in 49 commonly consumed Brassica vegetable samples in the United States. It is believed that this work introduces a new way of fast data processing and interpretation for qualitative and quantitative analyses of glucosinolates, where great efficacy was improved in comparison to identification manually.

Bioavailability and in vivo metabolism of intact glucosinolates

Health benefits associated with consumption of cruciferous vegetables have received considerable attention with a hitherto focus on the role and bioactivity of glucosinolate degradation products. We investigated the in vivo metabolism of intact glucosinolates by following their fate in digesta and in the endogenous metabolism in pigs. This is the first study to show an intact glucosinolate, sinalbin, being absorbed and transformed to a sinalbin metabolite in the liver by glucuronidation expectedly performed by liver phase II enzymes with subsequent excretion to the urine. From LC–MS/MS data we propose a structure of the sinalbin metabolite as containing two 4-oxybenzyl groups. Sinalbin and the metabolite were detected in plasma from the hepatic vein with a ratio of metabolite to sinalbin of approximately 12:1 after 2–4 hours. Induction of liver phase II enzymes by intact glucosinolates indicates that these also themselves are bioactive compounds with potential health risks or beneficial effects.

Glucosinolate Desulfation by the Phloem-Feeding Insect Bemisia tabaci

Glucosinolates are plant secondary defense metabolites confined nearly exclusively to the order Brassicales. Upon tissue rupture, glucosinolates are hydrolyzed to various bioactive breakdown products by the endogenous plant enzyme myrosinase. As the feeding of chewing insect herbivores is associated with plant tissue damage, these insects have developed several independent strategies for coping with the glucosinolate-myrosinase defense system. On the other hand, our knowledge of how phloem-feeding insects interact with the glucosinolate-myrosinase system is much more limited. In fact, phloem feeders might avoid contact with myrosinase altogether so their susceptibility to intoxication by glucosinolate hydrolysis products is unclear. Previous studies utilizing Arabidopsis thaliana plants accumulating high levels of aliphatic- or indolic-glucosinolates indicated that both glucosinolate groups have moderate negative effects on the reproductive performance of Bemisia tabaci, a generalist phloem-feeding insect. To get a deeper understanding of the interaction between B. tabaci and glucosinolate-defended plants, adults were allowed to feed on artificial diet containing intact glucosinolates or on Brussels sprout and A. thaliana plants, and their honeydew was analyzed for the presence of possible metabolites. We found that B. tabaci is capable of cleaving off the sulfate group of intact glucosinolates, producing desulfoglucosinolates that cannot be activated by myrosinases, a mechanism described to date only in several chewing insect herbivores. The presence of desulfated glucosinolates in the honeydew of a generalist phloem-feeder may indicate the necessity to detoxify glucosinolates, likely due to some level of cellular damage during feeding, which results in glucosinolate activation, or as a mechanism to circumvent the non-enzymatic breakdown of indolic glucosinolates.

Development and validation of a specific method to quantify intact glucosinolates in honey by LC–MS/MS

Glucosinolates (GLSs), which are secondary plant metabolites, have been scarcely investigated in honey. Recently, research has been conducted into the potential of GLSs as botanical biomarkers or as a parameter to establish honey freshness, but the employed methodologies were not specifically developed for intact GLSs quantification in honey. The aim of this study was to develop a new method to quantify twelve intact GLSs in honey, by using liquid chromatography with tandem mass spectrometry. An efficient extraction procedure was proposed; this involved dissolution of the honey with heated water, followed by solid-phase extraction with a weak anion exchange sorbent. Chromatography was performed on a 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). 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. The LOD and LOQ values were below 4μgkg−1 and 10μgkg−1, respectively. The proposed method was applied to analyze intact-GLSs in honey from different botanical origins. Four of the intact GLSs studied were identified at low concentration levels (<25μgkg−1) in several of the samples analyzed.

Development of a liquid chromatography–electrospray ionization–tandem mass spectrometry method for the simultaneous analysis of intact glucosinolates and isothiocyanates in Brassicaceae seeds and functional foods

A new high pressure liquid chromatography–electrospray ionization–tandem mass spectrometry method for the simultaneous determination of glucosinolates, as glucoraphanin and glucoerucin, and the corresponding isothiocyanates, as sulforaphane and erucin, was developed and applied to quantify these compounds in Eruca sativa defatted seed meals and enriched functional foods. The method involved solvent extraction, separation was achieved in gradient mode using water with 0.5% formic acid and acetonitrile with 0.5% formic acid and using a reverse phase C18 column. The electrospray ion source operated in negative and positive mode for the detection of glucosinolates and isothiocyanates, respectively, and the multiple reaction monitoring (MRM) was selected as acquisition mode. The method was validated following the ICH guidelines. Replicate experiments demonstrated a good accuracy (bias%<10%) and precision (CV%<10%). Detection limits and quantification limits are in the range of 1–400ng/mL for each analytes. Calibration curves were validated on concentration ranges from 0.05 to 50μg/mL.The method proved to be suitable for glucosinolates and isothiocyanates determination both in biomasses and in complex matrices such as food products enriched with glucosinolates, or nutraceutical bakery products.In addition, the developed method was applied to the simultaneous determination of glucosinolates and isothiocyanates in bakery product enriched with glucosinolates, to evaluate their thermal stability after different industrial processes from cultivation phases to consumer processing.

Naturally-Occurring Glucosinolates, Glucoraphanin and Glucoerucin, are Antagonists to Aryl Hydrocarbon Receptor as Their Chemopreventive Potency.

As a cytosolic transcription factor, the aryl hydrocarbon (Ah) receptor is involved in several patho- physiological events leading to immunosuppression and cancer; hence antagonists of the Ah receptor may possess chemoprevention properties. It is known to modulate carcinogen-metabolising enzymes, for instance the CYP1 family of cytochromes P450 and quinone reductase, both important in the biotransformation of many chemical carcinogens via regulating phase I and phase II enzyme systems. Utilising chemically-activated luciferase expression (CALUX) assay it was revealed that intact glucosinolates, glucoraphanin and glucoerucin, isolated from Brassica oleracea L. var. acephala sabellica and Eruca sativa ripe seeds, respectively, are such antagonists. Both glucosinolates were poor ligands for the Ah receptor; however, they effectively antagonised activation of the receptor by the avid ligand benzo[a]pyrene. Indeed, intact glucosinolate glucoraphanin was a more potent antagonist to the receptor than glucoerucin. It can be concluded that both glucosinolates effectively act as antagonists for the Ah receptor, and this may contribute to their established chemoprevention potency.

Analysis of Intact Glucosinolates in Beeswax by Liquid Chromatography Tandem Mass Spectrometry

A new method was developed to determine 12 intact glucosinolates (glucoiberin, glucoraphanin, glucoerucin, gluconapin, glucotropaeolin, glucobrassicin, gluconasturtiin, glucoalyssin, 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin, sinigrin) in beeswax, which is an authorized food additive in the European Union, by means of liquid chromatography tandem mass spectrometry with electrospray ionization. An efficient extraction procedure was proposed (average analyte recoveries were between 88 and 101 %); this involved the dissolution of melted beeswax with hexane, a solvent extraction using a mixture of ethanol and water (80:20, v/v), and a concentration step in a rotary evaporator. Chromatography was performed on a 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), at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification values were below 7 and 23 μg/kg, respectively. The proposed method was applied to analyze beeswax samples collected from organic apiaries.

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.