Pyrrolizidine alkaloids of Echium vulgare honey found in pure pollen

The pyrrolizidine alkaloids previously identified in floral honey attributed to Echium vulgare (Boraginaceae) have been detected (8000-14 000 ppm) in pure pollen collected from the anthers of Echium vulgare. Pyrrolizidine alkaloids and/or their N-oxides were isolated from the aqueous acid extracts of pollen by use of strong cation-exchange, solid-phase extraction and identified by liquid chromatographic/mass spectrometric (LCMS) analysis. The pyrrolizidine alkaloids in the pollen are present mainly as the N-oxides. In addition to seven previously described pyrrolizidine alkaloids and/or their N-oxides (echimidine, acetylechimidine, uplandicine, 9-O-angelylretronecine, echiuplatine, leptanthine, and echimiplatine), one unidentified (echivulgarine), but previously found in honey, and two previously undescribed (vulgarine and 7-O-acetylvulgarine) pyrrolizidine alkaloids and/or their N-oxides were identified in the pollen. Tentative structures for these unidentified pyrrolizidine alkaloids are proposed on the basis of the mass spectrometric data and biogenetic considerations. The implications of these results for identifying the source and subsequent concentrations of pyrrolizidine alkaloids in honeys and commercial bee pollen are briefly discussed.     

Solid-phase extraction and LC-MS analysis of pyrrolizidine alkaloids in honeys

Strong-cation-exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides from honey samples was followed by reduction of the N-oxides and subsequent analysis of total pyrrolizidine alkaloids using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. A limited survey of 63 preprocessing samples of honey, purposefully biased toward honeys attributed to floral sources known to produce pyrrolizidine alkaloids, demonstrated levels of pyrrolizidine alkaloids up to approximately 2000 parts per billion (ppb) in a sample attributed to Echium plantagineum. Up to 800 ppb pyrrolizidine alkaloids was detected in some honeys not attributed by the collector to any pyrrolizidine alkaloid-producing floral source. No pyrrolizidine alkaloids were detected in approximately 30% of the samples in this limited study, while some honeys showed the copresence of pyrrolizidine alkaloids from multiple floral sources such as E. plantagineum and Heliotropium europaeum. In addition, retail samples of blended honeys (with no labeling to suggest that pyrrolizidine alkaloid-producing floral sources were used in the blends) have been shown to contain up to approximately 250 ppb pyrrolizidine alkaloids.     

Hepatotoxic pyrrolizidine alkaloids in pollen and drying-related implications for commercial processing of bee pollen

Using HPLC-ESI-MS, several saturated and 1,2-dehydropyrrolizidine alkaloids were detected, mainly as their N-oxides, in fresh pollen collected from flowers of the pyrrolizidine alkaloid-producing plants Echium vulgare, E. plantagineum, Senecio jacobaea, S. ovatus, and Eupatorium cannabinum, and/or pollen loads from bees (bee pollen) that foraged on those plants. A major alkaloidal metabolite in S. ovatus was tentatively identified, using its mass spectrometric data and biogenic considerations, as the previously unreported, saturated alkaloid, 2-hydroxysarracine. Heating had very little effect on the 1,2-dehydropyrrolizidine alkaloids and their N-oxides from a variety of sources. Considered in conjunction with international concerns about the adverse effects of these alkaloids, the results strongly indicate a need for monitoring pollen supplies intended for human consumption, at least until conditions for processing and/or selection are clearly defined such as to significantly reduce the hepatotoxic (and potentially carcinogenic and genotoxic) pyrrolizidine alkaloid content of bee pollen.     

Analysis of herbal teas made from the leaves of comfrey (Symphytum officinale): reduction of N-oxides results in order of magnitude increases in the measurable concentration of pyrrolizidine alkaloids

OBJECTIVES: To determine the relative quantities of two hepatotoxic pyrrolizidine alkaloids, symphytine and echimidine, in teas prepared from comfrey leaves (Symphytum officinale), and to determine the potential contribution of the N-oxide forms of these alkaloids to levels of the parent alkaloids. DESIGN: Comfrey leaves were purchased from three commercial sources and used to prepare tea in a manner consistent with the methods used by consumers. An extraction scheme was devised for extraction of the alkaloids, and a gas chromatographic method was developed to quantify the two major alkaloids, symphytine and echimidine. Recognising that the N-oxide derivatives of these alkaloids have also been identified in comfrey preparations, chemical reduction was applied to determine the total quantities of the alkaloids as free bases and as N-oxide derivatives. RESULTS: The concentration of symphytine and echimidine varied considerably between teas prepared from leaves purchased from the different vendors of plant material. Moreover, a much higher concentration of symphytine was found in the tea when steps were included to reduce N-oxides prior to analysis. The treatment of pure symphytine with hot water did not generate the N-oxide derivative de novo. CONCLUSIONS: Since the pyrrolizidine alkaloids are known to be hepatotoxic, consumption of herbal teas made from comfrey leaves may be ill-advised. The concentration of pyrrolizidine alkaloids in such teas may be underestimated substantially unless the concentration of N-oxides is taken into consideration.     

Honey from plants containing pyrrolizidine alkaloids: a potential threat to health

Following scientific risk assessments, several countries have imposed strict regulations on herbal medicines containing 1,2-dehydro-pyrrolizidine alkaloids. Using published data on the plants used in honey production, pyrrolizidine alkaloid-containing plants are shown in this review to represent a significant source of honey worldwide. This observation, honey consumption data, reported levels of pyrrolizidine alkaloids in honeys, and consideration of tolerable exposure levels determined for pyrrolizidine alkaloids in herbal medicines, leads to the conclusion that some honey is a potential threat to health, especially for infants and fetuses, and further investigation is warranted.     

Analysis and occurrence of 14 sulfonamide antibacterials and chloramphenicol in honey by solid-phase extraction followed by LC/MS/MS analysis

A method was developed for the analysis of 14 sulfonamide antibiotics and chloramphenicol in honey. These antibiotics have been banned for use in food-producing animals; yet, their residues were found in many samples, illustrating the need for a multiresidue analysis for these antibiotics in honey. The method described here uses an acid hydrolysis step to liberate the sugar-bound sulfonamides followed by a solid-phase extraction to remove potential interferences. Analysis was by liquid chromatography--electrospray ionization--tandem mass spectrometry in negative mode for all 15 analytes. This MRM method generated two structurally significant transitions per compound, and it was designed to conform to U.S. Food and Drug Administration MS confirmation guidelines. It also provides 4-EU identification points. One hundred sixteen samples from 25 countries were analyzed, and 38% were found to contain at least one target antimicrobial. Five different target compounds were found in honey from 13 different countries.     

LC-MS/MS analysis of neonicotinoid insecticides in honey: methodology and residue findings in Austrian honeys

An analytical method for the simultaneous determination of residues of eight neonicotinoid insecticides and two metabolites in honey using LC-MS/MS was developed and validated. Two approaches of sample preparation were investigated, with the final method involving acetonitrile extraction and subsequent cleanup by dispersive solid-phase extraction (QuEChERS type). Validation was based on quintuplicate analysis at three fortification levels and showed satisfactory recoveries (60-114%) and high precision (RSDs between 2.7 and 12.8%). Low limits of detection and quantification could be achieved for all analytes ranging from 0.6 to 5 μg/kg and from 2 to 10 μg/kg, respectively. Investigations of Austrian honey samples revealed the presence of acetamiprid, thiacloprid, and thiamethoxam residues in honey; however, no sample exceeded the maximum residue limits. On average, flower honey samples contained neonicotinoid residues in higher quantities compared to forest honey samples.     

Analysis of phenolic and other aromatic compounds in honeys by solid-phase microextraction followed by gas chromatography-mass spectrometry

The solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS) was used for the analysis of phenolic and other aromatic compounds in honey samples from different floral origin. Different parameters affecting the efficiency of the extraction, such as the type of the stationary phase of the fiber, NaCl and acetic acid addition, and extraction time, were optimized for the detection of the maximum number of compounds in the shortest analysis time. A total of 31 compounds were detected, with most of them identified and quantified by GC-MS. The principal component analysis (PCA) was applied to the data matrix; the results allowed for the differentiation between honeydew and nectar honeys on the basis of the salicylic acid concentration. It was found that this acid has a high contribution in the honeydew group (71.2-705.9 microg/100 g of honey) compared to the nectar honey group (0-47.6 microg/100 g of honey). The comparison of data in each honey group enabled us to characterize the floral source of some honeys using some aromatic compounds as markers.     

Determination and confirmation of nitrofuran residues in honey using LC-MS/MS

A method was developed for the determination and confirmation of furazolidone, nitrofurazone, furaltadone, and nitrofurantoin as their side-chain residues in honey using liquid chromatography-tandem mass spectrometry (LC-MS/MS). An initial solid-phase extraction cleanup of the honey samples was followed by overnight hydrolysis and derivatization of the nitrofuran side-chain residues with 2-nitrobenzaldehyde. After pH adjustment and liquid-liquid extraction, the extracts were assayed by LC-MS/MS using electrospray ionization in the positive ion mode. The method was validated at concentrations ranging from 0.5 to 2.0 ppb with accuracies of 92-103% and coefficients of variation of < or =10%. The lowest calibration standard used (0.25 ppb) was defined as the limit of quantitation for all four nitrofuran side-chain residues. The extracts and standards were also used for confirmatory purposes. Honey from dosed beehives was assayed to study the stability of the nitrofuran residues and to demonstrate the effectiveness of the method.     

Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures

The usefulness of the solvent mixture dichloromethane/methanol for lipid extraction and the determination of lipid classes and fatty acids in samples of different natures was conducted. Two different extraction methods were compared, one containing chloroform/methanol, another containing dichloromethane/methanol. Total lipid extraction showed some minor differences but no variation in the lipid classes. Regarding the fatty acid profile, in Echium virescens seeds, 17 major fatty acids could be identified and quantified, and all were equally extracted when either solvent system was employed. In Echium acanthocarpum hairy roots, 17 major fatty acids were quantified, showing some statistical differences for one cell line in favor of chloroform. The data obtained from the liquid nutrient medium were also comparable. The cod roe sample showed 31 major fatty acids, showing no statistical differences between the two solvent systems. Contrarily, the CH 2Cl 2 method was able to extract 31 main fatty acids found in European seabass dorsal muscle more efficiently than the CHCl 3 method. The results indicate that, for lipid extraction and fatty acid assessment, dichloromethane/methanol can readily replace the commonly employed chloroform/methanol, thus avoiding the major health, security, and regulatory problems associated with the use of chloroform.     

Validation of an analytical methodology for determination of oxytetracycline and tetracycline residues in honey by HPLC with fluorescence detection

An analytical method for the determination of OTC and TC residues in honey was developed. Sample treatment involves an extraction in EDTA-McIlvaine buffer, followed by a solid-phase cleanup step. With regard to the cleanup procedure, different SPE cartridges were evaluated and the results presented. The method was validated according to the guidelines laid down by the 2002/657/EC European Decision parameters: decision limit (Cc alpha) and detection capability (CC beta) were 20 and 21 microg/Kg and 49 and 50 microg/Kg for OTC and TC, respectively, and recoveries of OTC and TC from spiked samples, at three fortification levels, were higher than 87% for both compounds. The analytical method was applied to 57 honey samples.     

Determination of low levels of perchlorate in lettuce and spinach using ion chromatography-electrospray ionization mass spectrometry (IC-ESI-MS)

A sample preparation method was developed to quantify environmentally relevant (low micrograms per liter) concentrations of perchlorate (ClO4(-)) in leafy vegetables using IC-ESI-MS. Lettuce and spinach were macerated, centrifuged, and filtered, and the aqueous extracts were rendered water-clear using a one-step solid-phase extraction method. Total time for extraction and sample preparation was 6 h. Ion suppression was demonstrated and was likely due to unknown organics still present in the extract solution after cleanup. However, this interference was readily eliminated using a Cl(18)O4(-) internal standard at 1 microg/L in all standards and samples. Hydroponically grown perchlorate-free butterhead lettuce was spiked to either 10.3 or 37.7 microg/kg of fresh weight (FW), and recoveries were between 91 and 98% and between 93 and 101%, respectively. Five types of lettuce and spinach from a local grocery store were then analyzed; they contained from 0.6 to 6.4 microg/kg of FW. Spike recoveries using the store-bought samples ranged from 89 to 100%. The method detection limit for perchlorate in plant extracts is 40 ng/L, and the corresponding minimum reporting limit is 200 ng/L or 0.8 microg/kg of FW.     

Classification and characterization of manuka honeys based on phenolic compounds and methylglyoxal

Manuka honey from New Zealand is often considered to be a medicinal product of special value due to its high level of antimicrobial activity. Therefore, the distinct authentication of its botanical origin is of great importance. Aside from the common pollen analysis, it is in this respect particularly the analysis of the phenolic acids, flavonoids, and norisoprenoids that is described as useful. In the present study, numerous manuka honeys were analyzed by UPLC-PDA-MS/MS after solid-phase extraction and compared to other kinds of honey to define marker substances characteristic for manuka honeys. The PDA profiles obtained differed markedly from each other so that the individual honey samples could be assigned to three groups. For the honeys of group 1 the comparably high concentrations of 4-hydroxybenzoic acid, dehydrovomifoliol, and benzoic acid proved to be typical, whereas the profiles of group 2 showed high kojic acid and 2-methoxybenzoic acid intensities. The manuka honeys of group 3, on the other hand, yielded high amounts of syringic acid, 4-methoxyphenyllactic acid, and methyl syringate. Furthermore, the comprehensive comparison of manuka honeys to other unifloral honeys revealed that especially kojic acid, 5-methyl-3-furancarboxylic acid, leptosin, unedone, 2-methoxybenzoic acid, 4-methoxyphenyllactic acid, 3-hydroxy-1-(2-methoxyphenyl)penta-1,4-dione, and methyl syringate were useful for distinguishing manuka honeys from the other kinds of investigated honeys. Moreover, kojic acid, unedone, 5-methyl-3-furancarboxylic acid, 3-hydroxy-1-(2-methoxyphenyl)penta-1,4-dione, and lumichrome were identified in manuka honey for the first time.     

Evaluation of residues of essential oil components in honey after different anti-varroa treatments

Apiary trials on the use of three different treatments (Apilife Var, thymol solution in olive oil, and thymol solution in ethanol) for the control of Varroa destructor were conducted in Aragon (northeastern Spain). For the evaluation of the presence of residues of these treatments in honey an analytical method was developed. The method is applied to analyze honey samples before and after treatments with the acaricides mentioned. A solid-phase extraction on trifunctional silane SPE C18 cartridge and gas chromatography separation using a flame ionization detector allow reliable and precise determination of residues of thymol, menthol, eucalyptol, and camphor in honey. The results indicate that camphor is present in only low concentrations, residues of eucalyptol or menthol were not found at all, and only thymol left residues in high concentrations. Residues of thymol found in honey collected from the beehives ranged from 0.75 to 8.20 microg/g for Apilife Var, from 0.03 to 6.30 microg/g for thymol solution in olive oil, and from 0.05 to 6.20 microg/g for thymol solution in ethanol. Even so, natural treatments can be considered to be good alternatives for synthetic acaricides, especially because they do not represent a sanitary risk.     

Development of enzyme-linked immunosorbent assays for the hepatotoxic alkaloids riddelliine and riddelliine N-oxide

Pyrrolizidine alkaloid-containing plants are widely distributed throughout the world and are particularly common in the genus Senecio. The structural types and concentrations of the alkaloids vary among plant species. In addition, within a species of plant, concentrations vary with environment and location. Many pyrrolizidine alkaloids are toxic and cause poisoning in livestock and in humans. Rapid, sensitive, and specific diagnostic techniques are needed to identify poisoned animals and to determine the particular plants and conditions under which livestock are likely to be poisoned. In this study, two competitive inhibition enzyme-linked immunosorbent assays for riddelliine, riddelliine N-oxide, and other closely related pyrrolizidine alkaloids were developed using polyclonal antibodies. One assay is class specific toward the free base forms of the pyrrolizidine alkaloids; the other assay showed cross-reactivity to both the free base and N-oxide forms of the alkaloids. The assay with the lowest limit of detection had an I(50) of 803.9 pg with a limit of detection of 47.5 pg for riddelliine. Spike and recovery studies for riddelliine in bovine blood ranged from 45 to 74%. The assay that showed cross-reactivity between the N-oxide and free base forms of the pyrrolizidine alkaloids allowed estimation of the total pyrrolizidine alkaloid content in Senecio riddellii in admixture with alfalfa. These findings suggest that these techniques will be excellent tools to diagnose poisoned animals and identify highly toxic plants.     

Assessment of pesticide residues in honey samples from portugal and Spain

Fifty samples of honey collected from local markets of Portugal and Spain during year 2002 were analyzed for 42 organochlorine, carbamate, and organophosphorus pesticide residues. An analytical procedure based on solid-phase extraction with octadecyl sorbent followed by gas chromatography-mass spectrometry (GC-MS), for organochlorines, and by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS), for organophosphorus and carbamates, has been developed. Recoveries of spiked samples ranged from 73 to 98%, except for dimethoate (40%), with relative standard deviations from 3 to16% in terms of repeatability, and from 6 to 19% in terms of reproducibility. Limits of quantification were from 0.003 to 0.1 mg kg(-)(1). Most of the pesticides found in honey were organochlorines. Among them, gamma-HCH was the most frequently detected in 50% of the samples, followed by HCB in 32% of the samples and the other isomers of HCH (alpha-HCH and beta-HCH) in 28 and 26% of the samples, respectively. Residues of DDT and their metabolites were detected in 20% of the samples. Of the studied carbamates, both methiocarb and carbofuran were detected in 10% of the samples, pirimicarb in 4% and carbaryl in 2%. The only organophosphorus pesticides found were heptenophos in 16%, methidathion in 4%, and parathion methyl in 2% of honey samples. Results indicate that Portuguese honeys were more contaminated than Spanish ones. However, honey consumers of both countries should not be concerned about the amounts of pesticide residues found in honeys available on the market.     

Development and validation of an optical SPR biosensor assay for tylosin residues in honey

In recent years there has been an increase in the use of tylosin in apiculture as bacterial brood diseases become resistant to oxytetracycline. Confirmatory mass spectrometry based methods have been developed but up until now there has been no complementary screening method available capable of sub 10 microg kg(-1) detection limits. In this paper the development and validation of a screening method using optical biosensor technology is presented. The honey was first dissolved in a phosphate buffer and following solid-phase extraction (SPE) cleanup was analyzed using a Biacore Q instrument. Using the criteria specified in European Commission Decision 2002/657/EC for qualitative screening methods, the detection capability (CCbeta) of the method was determined to be 2.5 microg kg(-)(1). Honey samples containing trace residue levels of tylosin were analyzed by both the biosensor screening method and a LC-MS/MS confirmatory procedure; the results were in good agreement.     

Multiclass determination and confirmation of antibiotic residues in honey using LC-MS/MS

A multiclass method has been developed for the determination and confirmation in honey of tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline), fluoroquinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, and sarafloxacin), macrolides (tylosin), lincosamides (lincomycin), aminoglycosides (streptomycin), sulfonamides (sulfathiazole), phenicols (chloramphenicol), and fumagillin residues using liquid chromatography tandem mass spectrometry (LC-MS/MS). Erythromycin (a macrolide) and monensin (an ionophore) can be detected and confirmed but not quantitated. Honey samples (approximately 2 g) are dissolved in 10 mL of water and centrifuged. An aliquot of the supernatant is used to determine streptomycin. The remaining supernatant is filtered through a fine-mesh nylon fabric and cleaned up by solid phase extraction. After solvent evaporation and sample reconstitution, 15 antibiotics are assayed by LC-MS/MS using electrospray ionization (ESI) in positive ion mode. Afterward, chloramphenicol is assayed using ESI in negative ion mode. The method has been validated at the low part per billion levels for most of the drugs with accuracies between 65 and 104% and coefficients of variation less than 17%. The evaluation of matrix effects caused by honey of different floral origin is presented.     

Rapid quantification of proanthocyanidins (condensed tannins) with a continuous flow analyzer

Proanthocyanidins (condensed tannins) frequently need to be quantified in large numbers of samples in food, plant, and environmental studies. An automated colorimetric method to quantify proanthocyanidins with sulfuric acid (H(2)SO(4)) was therefore developed for use in a continuous flow analyzer. Assay conditions were optimized using 50% methanol extracts of paper birch, sugar maple, and quaking aspen leaves. Short extraction times and centrifugation of samples prevented proanthocyanidin degradation that otherwise occurred in 50% methanol extracts of aspen leaves. Extraction of birch and maple proanthocyanidins with 50% methanol was comparable to or better than that with 70% acetone. Proanthocyanidin levels in aspen were lower when extracted with aqueous methanol, but relative differences among samples were consistent with those found in aqueous acetone extracts. Results from the automated sulfuric acid assay were highly correlated with those of the conventional BuOH-HCl method for proanthocyanidins and, except for birch, with the Folin--Denis assay for total phenolics. This new technique significantly improves assay processing rate and repeatability compared to conventional colorimetric proanthocyanidin assays.     

Gas chromatographic organic acid profiling analysis of brandies and whiskeys for pattern recognition analysis

An efficient gas chromatographic profiling and pattern recognition method is described for brandy and whiskey samples according to their organic acid contents. It involves solid-phase extraction of organic acids using Chromosorb P with subsequent conversion to stable tert-butyldimethylsilyl derivatives for the direct analysis by capillary column gas chromatography and gas chromatography-mass spectrometry. A total of 12 organic acids were reproducibly identified in liquor samples (1 mL). When the GC profiles were simplified to their retention index spectra, characteristic patterns were obtained for each liquor sample as well as for each group average. Stepwise discriminant analysis provided star symbols characteristic for each liquor sample and group average. As expected, canonical discriminant analysis correctly classified 23 liquor samples studied into two groups of either brandy or whiskey.