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Pyrrolizidine alkaloids from Echium vulgare in honey originate primarily from floral nectar Matteo A. Lucchetti, Gaétan Glauser, Verena Kilchenmann, Arne Dübecke, Gudrun Beckh, Christophe Praz, and Christina Kast J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02320 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016
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Pyrrolizidine alkaloids from Echium vulgare in honey originate
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primarily from floral nectar
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Matteo A. Lucchetti , Gaetan Glauser , Verena Kilchenmann , Arne Dübecke , Gudrun Beckh ,
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Christophe Praz2, Christina Kast1*
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1
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Rue Emile-Argand 11, 2000 Neuchâtel, Switzerland.
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1,2
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Agroscope, Swiss Bee Research Centre, Schwarzenburgstrasse 161, 3003 Bern, Switzerland. Institute of Biology, Laboratory of Fundamental and Applied Research in Chemical Ecology (FARCE), University of Neuchâtel,
Neuchâtel Platform of Analytical Chemistry, University of Neuchâtel, Rue Emile-Argand 11, 2000 Neuchâtel, Switzerland. Quality Services International GmbH (QSI), Flughafendamm 9a, 28199 Bremen, Germany.
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*Corresponding author: Email:
[email protected] 11
Abstract
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Pyrrolizidine alkaloids (PAs) in honey can be a potential human health risk. So far, it
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has remained unclear whether PAs in honey originate from pollen or floral nectar. We
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obtained honey, nectar and plant pollen from two observation sites where Echium
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vulgare L. was naturally abundant. The PA concentration of honey was determined
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by targeted analysis using a high pressure liquid chromatography-mass spectrometry
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system (HPLC-MS/MS), allowing the quantification of six different PAs and PA-N-
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oxides present in E. vulgare. Echium-type PAs were detected up to 0.153 µg/g in
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honey. Nectar and plant pollen were analyzed by non-targeted analysis using ultra-
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high pressure liquid chromatography-high resolution-mass spectrometry (UHPLC-
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HR-MS), allowing the detection of 10 alkaloids in small size samples. Echium-type
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PAs were detected between 0.3 - 95.1 µg/g in nectar and 500 - 35000 µg/g in plant
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pollen. The PA composition in nectar and plant pollen was compared to the
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composition in honey. Echimidine (+N-oxide) was the main alkaloid detected in
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honey and nectar samples, while echivulgarine (+N-oxide) was the main PA found in
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plant pollen. These results suggest that nectar contributes more significantly to PA
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contamination in honey than plant pollen.
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Keywords: Pyrrolizidine alkaloids, Echium vulgare, honey, nectar, plant pollen, high
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pressure liquid chromatography-mass spectrometry (HPLC-MS/MS), ultrahigh
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pressure liquid chromatography-high resolution mass spectrometry (UHPLC-HR-MS).
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1.1
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Pyrrolizidine Alkaloids (PAs) are toxic compounds produced by plants as a chemical
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defense against herbivores.1,2 Many unrelated plant species produce PAs, and some
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of them are particularly abundant in European agro-ecosystems. These PA-
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containing plants mainly belong to the Asteraceae (Senecioneae and Eupatorieae
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tribes), Boraginaceae (all genera), and Fabaceae (genus Crotalaria) families.3,4 PAs
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may get into the food chain when food products are either contaminated with or
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derived from PA-containing plant tissues.5-7 PAs can occur as N-oxides or as free-
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base/tertiary forms. These two forms are both hepatotoxic and genotoxic.8,9 In plants,
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the N-oxides are found in higher concentrations than the corresponding free-bases
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(tertiary PAs).1,10 Acute poisoning or chronic exposure to PAs mostly affects liver
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function, since PAs are activated by nucleophilic compounds through the liver’s
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detoxification enzymes.6,11-13 The chronic intake of low PA-levels in food can lead to
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liver cirrhosis and cancer.14 Legal limits for PAs in food have not yet been established
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in the European Union or in Switzerland. However, the German Federal Institute for
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Risk Assessment (BfR) recommends an intake of not more than 0.007 µg of 1.2-
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unsaturated PAs per day per kg bodyweight.15
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Honey is one of the best studied food products with respect to PA contamination.
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When honeybees collect nectar and plant pollen from PA-containing plants, PAs are
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transferred into bee products such as honey or bee-collected pollen.16,17 PAs have
Introduction
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been detected in honey samples from various geographical and botanical origins.4,18-
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by the BfR have been reported in monofloral honeys from Echium vulgare or E.
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plantagineum 28-32 and from Senecio jacobaea.31,33,34
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Honey is mostly composed of concentrated nectar and contains only traces of pollen.
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Therefore, PAs contained in nectar constitute an important potential source of PAs in
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honeys. However, the concentration of secondary compounds may be considerably
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higher in plant pollen than in nectar,35,36,37 and some pollen types contain particularly
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high amounts of PAs.17 Consequently, it remains unclear whether the PA content in
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nectar is high enough to substantially contaminate honey,38 and, more generally,
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whether the PAs in honey predominantly originate from pollen or nectar.39,40
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Prior research has suggested pollen as the major source of PA contamination in
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honey.14,17 Contamination of honey could be caused by the liberation of PAs from
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pollen.38 Lastly, experiments with plant pollen from Senecio vernalis added to PA-free
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honey have suggested that contamination of honey may occur through diffusion of
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PAs from pollen into honey.40
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Unraveling the entry mechanism by which PAs contaminate honey is important for
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reducing risks associated with PA-containing bee products.
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Two approaches can be used to examine the pathway from the different plant tissues
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into bee products. First, quantitative analyses of nectar, pollen, and honey may help
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determine which plant part is the main contributor to the total PAs found in honey.
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Second, differences in the PA composition (relative abundance of different PAs)
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found in nectar can be compared to that found in pollen. This information can be
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used to determine whether the PA composition in honey more closely matches that
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of nectar or pollen. In the present study, we performed qualitative and quantitative
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analysis of the different alkaloids found in Echium nectar and pollen.
PA concentrations of up to two orders of magnitudes over the limits recommended
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We selected Echium vulgare as a model to study the pathway by which PAs are
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transferred into honey. This plant species is the only species of the genus Echium
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vulgare regularly found in Switzerland. It is widely distributed in Europe and has been
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previously described as a major source of PA contamination of European honeys.19,21
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We chose two observation sites where E. vulgare was blooming during the bee
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season. These sites were located in two different climatic regions, one located to the
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north and the other to the south of the Alps.
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1.2 Material and Methods
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Chemical reagents
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The echimidine used in this study was obtained from Phytolab (Vestenbergsgreuth,
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Germany), while the heliotrine was from Latoxan (Valence, France). For plant
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extraction, milli-Q water was used. Formic acid and glass beads (Ø 2 mm) were
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purchased from Sigma-Aldrich (Buchs, Switzerland). HPLC grade methanol was
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purchased from Merck (Dietikon, Switzerland). Cyclohexane (> 98% purity), sulphuric
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acid, and ammonia were purchased from Merck Chemicals (Darmstadt, Germany).
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The solvents and additives used for LC-MS were water, acetonitrile, and ULC-MS
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grade formic acid acquired from Biosolve (Valkenswaard, Netherlands).
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Observation sites
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We selected two observation sites where Echium plants were abundant around bee
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colonies. The first observation site was located north of the Alps near Basel, close to
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the border between Switzerland and France (hereafter Basel). The other observation
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site was located close to Italy, along the southern flank of the Alps in the Verzasca
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valley (hereafter Verzasca). At the Verzasca site, two beekeepers participated to the
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project (hereafter Verzasca 1 and Verzasca 2). The aerial distance between these
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two apiaries was approximately 400 meters.
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Honey and plant sample collection
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Honeys: In total, four samples of honey were included from Basel and six from
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Verzasca. In Basel, the honeys were harvested on 8 June and 27 July 2012, as well
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as on 29 June and 8 August 2013. In Verzasca 1, the honeys were harvested on 1
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August 2012, 2 August 2013, and 1 August 2014, while in Verzasca 2 they were
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harvested on 30 July 2012, 2 August 2013, and 1 August 2014. Eight additional
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honey samples were obtained from apiaries in diverse regions of Switzerland. All of
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the additional honey samples were produced between 2009 and 2011. Plant
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material: In Basel, E. vulgare was in blossom during June and July, while in the
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Verzasca valley E. vulgare was in blossom from June until August. Samples of nectar
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and plant pollen from E. vulgare were collected at the two observation sites. Samples
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were only collected under dry weather conditions to avoid wash-out of plant pollen
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from the anthers and dilution of nectar by the rain. In Basel, the samples were
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collected on 18 June and 4 July 2013 and on 19 June and 27 June 2014. In
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Verzasca, the samples were collected on 29 June and 17 July 2013 and on 6 July
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and 18 July 2014. In total, 20 nectar samples from Basel (n=10 in 2013 and n=10 in
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2014), 16 nectar samples from Verzasca (n=7 in 2013 and n=9 in 2014), 14 plant
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pollen samples from Basel (n=5 in 2013 and n=9 in 2014) and 13 plant pollen
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samples from Verzasca (n=4 in 2013 and n=9 in 2014) were collected. On the day
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before any given sample collection, plants were tightly bagged with a fine mesh, and
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a layer of insect glue was spread around the lower part of the stem in order to
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prevent insect visits. Nectar was collected using a Pasteur pipette previously
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elongated to a capillary on a flame. The pipette was directly placed into the corolla,
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carefully avoiding any disruption of floral tissues. In 2013, pollen from anthers of
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Echium flowers (plant pollen) was collected with metal forceps. Plant pollen was
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carefully removed from the surface of the anthers in order to prevent contamination
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of the pollen with other flower parts, especially the anthers. This procedure yielded
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low amounts of plant pollen. In order to facilitate collection and avoid contamination
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with other plant parts, plant pollen was collected in 2014 by immersing the stamens
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into cyclohexane.38 The cyclohexane was subsequently evaporated. For comparison,
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two plant pollen samples were collected from the same plant using both methods
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(forceps and cyclohexane). Since the two samples gave comparable results (data not
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shown), we concluded that the cyclohexane did not wash out PAs from the pollen,
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and hence that both collection methods would yield similar results. All samples were
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kept on dry ice during collection and subsequently stored at -80°C until extraction.
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Sample preparation of honey for quantification of PAs with LC-MS/MS
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Honey samples were prepared as described in Dübecke et al.19 Since PA-N-oxides
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are polar organic compounds with basic characteristics, they are soluble in polar
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organic solvents or in mixtures of solvents and acidified water. 10 g of honey,
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together with 100 ng heliotrine as internal standard and 30 mL of 0.05M sulphuric
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acid were vigorously shaken for 20 min. Samples were then filtered overnight using a
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2 mm mesh to remove particles that could block the solid-phase extraction. Clean-up
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was conducted using SPE-SCX Cartridges (Varian) washed previously with methanol
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and conditioned with 9 mL of 0.05M sulphuric acid. Samples were loaded onto the
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column, washed with 9 mL of deionized water, eluted into a glass vial using
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ammoniated methanol,18 and dried at 40°C in an ambient air stream. Samples were
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then reconstituted in 1 mL deionized water, shaken vigorously, and filtered into a 2
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mL glass vial using a 0.45 µm syringe filter.
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The PA concentration was determined by targeted analysis using a HPLC-MS/MS-
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system as described in Dübecke et al.,19 allowing the detection of six different PAs or
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PA-N-oxides (echimidine, echimidine-N-oxide, acetylechimidine, acetylechimidine-N-
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oxide, echivulgarine and echivulgarine-N-oxide) commonly found in E. vulgare.17 The
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total PA concentration was calculated as the sum of the six different PAs. LC-MS/MS
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analysis was performed using an HTC PAL autosampler of CTC Analytics AG, a
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Shimadzu LC-system with a Thermo Hypersil Gold C18 column (50 x 2.1 mm, 1.9 µm
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particle size) and an Applied Biosystems API4000 QTRAP triple quadrupole mass
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spectrometer. Concentrations were corrected against the recovery of the internal
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standard. For quantification, external calibration was performed using echimidine as
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the standard. The limit of quantitation (LOQ) for echimidine in honey was 1 ng/g. As
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no further reference standards were available, echimidine-N-oxide, acetylechimidine,
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acetylechimidine-N-oxide, echivulgarine, and echivulgarine-N-oxide were indirectly
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quantified using the calibration of echimidine, assuming the same response factor
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and thus the same LOQ. A linear range was achieved from 0.5 to 100 ng/mL for the
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echimidine standard. Recovery of echimidine near the LOQ was 97%. Repeatability
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was 5.4% as determined with six independent sample preparations measured by the
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same person on the same day.
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Extraction of PAs from nectar and plant pollen, and UHPLC-HRMS analysis
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Nectar: 5 µL of nectar was directly transferred into a glass vial containing a 200 µL
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insert and diluted 10 times with the extraction solvent (70% methanol, 29.5% ultra-
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pure water and 0.5% formic acid, v/v). Plant pollen: 1 mg of plant pollen was
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accurately weighed using a microbalance scale (Mettler Toledo), mixed with 100 µL
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of extraction solvent as described above and transferred into a 2 mL Eppendorf tube.
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Five glass beads were added and the tube was vigorously shaken at 30 Hz for 4 min
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to disrupt the pollen structure and to extract the PAs. Following centrifugation (18400
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g, 4 min), 5 µL of the supernatant was transferred into a glass vial containing a 200
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µL insert and diluted 10 times with the extraction solvent.
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Non-targeted analysis using the UHPLC-HR-MS system was carried out for the
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detection and quantification of various alkaloids found in E. vulgare (echimidine,
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echimidine-N-oxide,
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echimidine-N-oxide, acetylvulgarine, acetylvulgarine-N-oxide, echivulgarine and
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echivulgarine-N-oxide17). In brief, separation of the alkaloids was performed on an
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Acquity BEH C18 column (50 x 2.1 mm i.d., 1.7 µm particle size, Waters), using an
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Acquity UHPLCTM system (Waters) coupled to a Synapt G2 QTOF mass
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spectrometer (Waters). A binary gradient was performed at a flow rate of 0.4 mL min-
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1
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formic acid in acetonitrile (solvent B). The gradient program was as follows: 0-4 min
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5-40% B, 4-6 min 40-100% B, 6-9 min 100% B, 9.1-10.5 min 5% B. The temperature
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of the column was maintained at 30°C and that of the autosampler at 25°C. Injection
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volume was 1 µL. The QTOF operated in electrospray positive mode over a mass
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range of 50-600 Da. MS conditions were: Capillary voltage +2800 V, cone voltage
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+30 V, source temperature 120°C, desolvation gas temperature and flow 350°C and
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800 L/h, respectively, and scan time 0.4 sec. A leucine-enkephaline solution at 400
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ng/mL was infused throughout the analysis to ensure high mass accuracy (
