Research article Received: 18 February 2013
Revised: 27 June 2013
Accepted: 5 July 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3251
A rapid and highly specific method to evaluate the presence of pyrrolizidine alkaloids in Borago officinalis seed oil Giulio Vacillotto,a Donata Favretto,b Roberta Seraglia,a* Rita Pagiotti,c Pietro Traldia and Luisa Mattolid Pyrrolizidine alkaloids (PAs) are complex molecules, present in plants as free bases and N-oxides. They are known for their hepatotoxicity, and consequently there is a health risk associated with the use of medicinal herbs that contain PAs. Unfortunately, there is no international regulation of PAs in foods, unlike those for herbs and medicines: in particular, for herbal preparation or herbal extracts, the total PA content must not exceed 1 μg/kg or 1 μg/l, respectively. Borago officinalis seed oil is a source of γ-linolenic acid, and its use is increased in both pharmaceutical and health food industries. Even if studies based on gas chromatography and TLC methods showed that PAs are not co-extracted with oil, the development of a rapid and sensitive method able to evaluate the presence of PAs in commercially available products is surely of interest. The presence of PAs in a commercially available Borago officinalis seed oil was tested either in the oil sample diluted with tetrahydrofuran/methanol (MeOH)/H2O (85/10/5 v:v:v) or after extraction with MeOH/H2O (50/50 v:v) solution The samples were analysed by electrospray ionization in positive ion mode and in high mass resolution (60 000) conditions. In both cases to evaluate the effectiveness of the method, spiking experiments were performed adding known amount of two PA standards to the borage seed oil. A limit of detection in the order of 200 ppt was determined for these two compounds, strongly analogous to Borago officinalis seed oil PAs. Consequently, if present, PAs level in Borago officinalis seed oil must lower than 200 ppt. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Pyrrolizidine alkaloids; Borago officinalis seed oil; high resolution mass spectrometry; tandem mass spectrometry; electrospray ionization
Introduction
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Pyrrolizidine alkaloids (PAs) are complex molecules, consisting of two fused five-membered rings jointed by a single nitrogen atom at position 4 to form a heterocyclic nucleus (see Fig. 1). PAs are plant products produced as secondary metabolites[1] and are sometimes toxic to animals when eaten.[2] Their biological role in plants is quite unknown, but it has been supposed that PAs have evolved as a defense mechanism against unadapted generalist herbivores,[3] while specialist herbivores use this class of compounds for recognizing their food plant, acting as an oviposition stimulant[4] and as feeding stimulant.[5] PAs are present in plants as free bases and as N-oxides (PANOs). More than 600 different structure of PAs and PANOs were identified in over 6000 plant species,[3] and they are mainly present in the families Boraginaceae (all plants), Asteraceae (tribes Senecionaceaea and Eupotoriceae), Orchidaceaea (nine genera) and Fabaceae (mainly the genus Crotalaria).[6] PAs are metabolized in the liver, producing toxic metabolites related to their structure and to the presence of species-specific liver enzymes.[7,8] This toxicity is considered responsible for veno-occlusive liver disease,[9] consisting in the obstruction of the small veins bringing blood from the liver back to the heart. Aside from ingesting the plant directly, PAs may be assumed by eating honey[10–13] and eggs[14] and by drinking milk,[15] produced by animals which have consumed PAs-containing plants. Of concern is the health risk associated with the use of
J. Mass Spectrom. 2013, 48, 1078–1082
medicinal herbs that contain PAs, notably borage leaf, comfrey and colt’s foot in the West, and some Chinese medicinal herbs.[16,17] The European Food Safety Authority was asked by the European Commission to deliver a scientific opinion on PAs in food and feed. The Panel on Contaminants in the Food Chain (CONTAM Panel) performed estimates of both acute and chronic exposure to PAs through honey for three different age groups. Although there might be other sources of PAs exposure, due to lack of data, the CONTAM Panel was not able to quantify dietary exposure from food other than honey. Based on the present knowledge, the CONTAM Panel concluded that 1,2-unsaturated PAs may act as genotoxic carcinogens in humans and supports ongoing efforts to collect more data on the PAs identified and potentially found in feed and relevant foods, including milk, eggs
* Correspondence to: Roberta Seraglia, CNR-ISTM, Corso Stati Uniti 4, 35127 Padova, Italy. E-mail: [email protected] a CNR-ISTM, Corso Stati Uniti 4, I35127 Padova Italy b Department of Molecular Medicine, University Hospital of Padova, I-35121 Padova Italy c Dipartimento di Biologia Vegetale Applicata, Università degli Studi di Perugia Perugia, Italy d Research Area, Aboca S.p.A. Società Agricola, loc. Aboca, Sansepolcro AR, Italy
Copyright © 2013 John Wiley & Sons, Ltd.
Method for evaluating PAs in Borago seed oil
Figure 1. General chemical structure of pyrrolizidine alkaloids and of Borago officinalis pyrrolizidine alkaloids.
J. Mass Spectrom. 2013, 48, 1078–1082
Experimentals Chemicals Tetrahydrofuran (THF), methanol (MeOH), chloroform and water were all HPLC grade and were purchased from Fluka (Milan, Italy).
Samples Borago officinalis oil was supplied by Aboca S.p.A.(Sansepolcro, Italy). Monocrotaline and Retrosine standards were purchased from Sigma–Aldrich (Milan, Italy).
Preparation of Borago officinalis oil sample for analysis. a) Dilution. Aliquots of Borago officinalis oil were diluted in THF/ MeOH/H2O (85:10:5 v:v:v) in ratios ranging from 1: 103 to 1: 106 w/w and vortex mixed for 60 s. In these conditions, a clear solution is obtained, without any phase separation. It is to note that the PAs concentration is necessarily reduced. b) Extraction. 1 ml of MeOH/H2O (50/50 v/v) was added to 250 μl of Borago officinalis oil in a conic, glass vial. After 60 s vortex mixing, the oil was extracted by inversion for 20 min and centrifuged at 13 000 rpm for 10 min. 900 μl of the clear supernatant, containing the PAs fraction, was transferred, and 100 μl of 1% formic acid was added.
Spiking experiments Due to the lack of commercially available reference standards of compounds 1–5, the alkaloids monocrotaline (6) and retrorsine (7) were used as model for the analytical procedures. They were dissolved in MeOH/H2O (50/50 v/v) at concentrations of 32.5 pg/ml, 325 pg/ml, 3.25 ng/ml, 32.5 ng/ml and 325 ng/ml. 100 μl of these solutions was added to a borage seed oil solution in THF/MeOH/H2O (85:10:5 v:v:v) containing 0.1 mg of oil. The solutions so obtained were directly infused into the mass spectrometer. Alternatively, compounds 6 and 7 were dissolved in CHCl3 at concentrations of 200 pg/ml, 2 ng/ml, 20ng/ml, 200ng/ml, 2 μg/ml and 20 μg/ml. 100 μl of these solutions was added to 1 g aliquots of borage oil. The oil aliquots were subsequently extracted as described above with MeOH/H2O.
Copyright © 2013 John Wiley & Sons, Ltd.
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and meat as well as in herbal dietary supplements prepared from PAs-containing plants. PAs are also found in the plant Borago officinalis[18–20](borage), which is an annual herbaceous plant native to Europe, North Africa and Asia Minor.[21] Most of borage PAs have a 1,2-unsaturation in the ring structure indicating hepatotoxicity[20] like amabiline[22] (1), lycopsamine[19] (2), intermidine[19](3) and the acetyl derivatives of lycopsamine and intermidine[19] (4,5) (Fig. 1). During the last ten years, the use of borage seed oil as a source of γ-linolenic acid (GLA) was increased in both pharmaceutical and health food industries. In fact, the oil extracted from the seed of Borago officinalis contains about 20% (w/w) of GLA.[23] Studies carried out by gas chromatography (GC) and TLC methods[18,24,25] showed that PAs are not co-extracted with the oil. These analytical methods can measure the PAs levels in the ppm range for GC[18,24] and in the ppb range for TLC[25] approaches. However, for using borage seed oil in pharmaceuticals, its PAs content must be determined to ensure the complete absence of these compounds. Consequently the development of a rapid and sensitive analytical procedure able to evaluate the presence of PAs in commercially available products is surely of interest. For the analysis of compounds at trace level ( 60 000 (FWHM as defined at m/z 400) and a scan time of 0.65 s. Mass calibration was performed according to the manufacturer’s guidelines using a manufacturer-defined mixture of sodium dodecyl sulphate, sodium taurocholate, the tetrapeptide MRFA (L-methionylarginyl-phenylalanyl-alanine acetate) and Ultramark 1621.
For the investigation on the most relevant alkaloids possibly present in Borago officinalis oil, the direct infusion of diluted oil samples has been employed. To this purpose, the oil was diluted in THF/MeOH/H2O (85:10:5 v:v:v) in the range 1:103- 1:106 and directly infused into the ESI source of the Orbitrap, working in full scan HRMS mode. The best results in terms of signal intensity and stability have been obtained with a dilution 1:104 and with a flow rate of 10 μl/min. As expected, it is particularly complex, and, considering that the ESI conditions employed are not suitable for activating any ‘in source’ fragmentation, it can be considered a fingerprint of the protonated molecular species present in the Borago officinalis oil. In particular, the highly complex ion clusters in the m/z range 800 – 1000 can be ascribed to Na+ adducts of triglycerides present in the oil. However, the direct injection of diluted oil sample does not give any information on the possible presence of the alkaloids typical of Borago officinalis. No peaks corresponding to the [M+H]+ ions of Amabiline (1, accurate mass: 283.1784), lycopsamine (2) and intermidine (3) (accurate mass: 299.1598) and the acetyl derivatives of licopsamine (4) and intermidine (5) (accurate mass: 341.1833) at m/z 284.1856, 300.1676 and 342.1911, respectively, were detected. These results could be due either to the real absence of the alkaloids or to the 1: 104 dilution step prior to analysis that would hamper the detection of components present in the oil at a very low concentration (e.g. at ppb level). In order to verify the latter hypothesis, the oil was extracted with MeOH/H2O (1:1 v:v) to obtain the hydrophilic fraction of the oil in which the PAs should be selectively extracted. Following the preparation reported in the experimental section, the extract so obtained was directly injected in the ESI source. The related spectrum shows again the complete lack in the ESI spectrum of the PAs-related peaks and the disappearance of the ions due the lipophilic species detected in THF/MeOH/H2O.
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Figure 3. Positive ion HRMS spectrum of Borago officinalis seed oil in THF/MeOH/H2O (85/10/5 v/v/v) spiked with 325 ppb of compounds 6 and 7.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2013, 48, 1078–1082
Method for evaluating PAs in Borago seed oil
+
Figure 6. MS/MS spectra of [M+H] ions of compounds 6 (a) and 7 (b), added at a concentration of 200 ppt to Borago officinalis seed oil extracted with MeOH/H2O (50/50 v/v).
Figure 4. Plots of concentration of compounds 6 and 7 versus absolute + abundance of their [M+H] ions (m/z 326.1604 for compound 6 and m/z 352.1760 for compound 7) obtained for: (a) dilution with THF/MeOH/H2O (85/10/5 v/v/v) and (b) extraction with MeOH/H2O (50/50 v/v).
+
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Figure 5. MS/MS spectra of [M+H] ions of compounds 6 (a) and 7 (b), added at a concentration of 32.5 ppb to Borago officinalis seed oil extracted with THF/MeOH/H2O (85/10/5 v/v/v).
Considering that compounds 1–5 are not commercially available, further experiments were carried out by spiking the oil with known, equimolar amounts of two standard alkaloids, respectively monocrotaline (6, MW 325.1511) and retrorsine (7, MW 351.1666). They both lead, in ESI conditions, to abundant [M+H]+ species at m/z 326.1589 and 352.1744, respectively. In order to increase the specificity of the measurements, their behaviour in collisional conditions was studied. The product ion spectra of [M+H]+ of the two alkaloids are reported in Fig. 2, in which the accurate mass values of the fragment ions are reported together with the chemical structures of monocrotaline (Fig. 2a) and retrorsine (Fig. 2b). The first spiking experiment has been performed by adding the standard solutions in the oil diluted in THF/MeOH/H2O, so to obtain standard concentrations in the range 32.5 ppb – 325 ppm. To evaluate the limit of detection (LOD) by direct infusion of the mixture in the ESI source, the presence of [M+H]+of 6 and 7 in high resolution, full scan mode, with a signal-to-noise ratio ≥ 3, was monitored. Even though the spectra are highly complex (see Fig. 3), the two diagnostic peaks are well detectable down to 325 ppb, that can be considered the LOD, as shown in the plot of Fig. 4a, related to the lower concentration used (32.5 ppm – 32.5 ppb). Alternatively to HRMS in full scan mode, an MS/MS approach was also undertook by using the LTQ portion of the Orbitrap. The results obtained are strictly analogous to those achieved in high resolution conditions, with an LOD value of 325 ppb. For the lowest standard concentration (32.5 ppb), the MS/MS spectrum becomes not specific, due to the presence of fragment ions originated from ionic species isobaric with the [M+H]+ ions of the two standard alkaloids (Fig. 5). Considering that a putative concentration of alkaloids in oil at 1 ppm level would become 100 ppt when the oil sample is diluted 1:104 in THF/MeOH/H2O prior to direct infusion, the LODs verified on standards (325 ppb) would not allow the identification of pyrrolyzidine alkaloids at such a low level in borage oil.
G. Vacillotto et al. In the second spiking experiment, the two standard alkaloids 6 and 7 have been added to the oil in a concentration range from 2 ppm to 20 ppt and extracted by MeOH/H2O. By direct analysis of the samples after extraction, in HRMS full scan mode, an LOD in the order of 200 ppt was obtained, as shown in the plot of Fig. 4b, related to the lower concentration used (100 ppb – 20 ppt). Similar LODs were also achieved by using the MS/MS scan approach on the MeOH/water extract, obtaining characteristic fragment products for the [M+H]+ of the two analytes spiked at 200 ppt (Fig. 6). The above described results, though obtained on different alkaloids of the pyrrolizidine family, reasonably suggest that amabiline, lycopsamine, intermidine and the acetyl derivatives of lycopsamine and intermidine, if present in the Borago officinalis oil under investigation, are at level lower than 200 ppt. This rapid, non-chromatographic approach turned out to be valuable for quality control of borage oil preparation. An extraction/enrichment step in the preparation of oil samples was demonstrated to be necessary to reach low LODs at the ppt level.
REFERENCES [1] T. Hartmann, C. Theuring, T. Beuerle, L. Ernst, M.S. Singer, E.A. Bernays. Acquired and partially de novo synthesized pyrrolidine alkaloids in two polyphagusarctiis and the alkaloid profiles of their larval food-plant. J. Chem. Ecol. 2004, 30, 229. [2] D.M. Holstege, J.N. Seiber, F.D. Galey. Rapid multiresidue screen for alkaloids in plant material and biological sample. J. Agricul. Food Chem. 1995, 43, 691. [3] L.J. Doorduin, K. Vrieling. A review of the phytochemical support for shifting defence hypothesis. Phytochem. Rev. 2011, 10, 99. [4] M. Màčel, K. Vrielin. Pyrrolizidine alkaloids as oviposition stimulants for the cinnabar moth, Tyriajacobaeae. J. Chem. Ecol. 2003, 29, 1435. [5] E.A. Bernays, T. Hartmann, R.F. Chapman. Gustatory responsiveness to pyrrolizidine alkaloids in the Senecio specialist, Tyriajacobaeae (Lepidoptera, Actiidae). Phys. Entomol. 2004, 29, 67. [6] D. Oder, T. Hartmann. Homospermidine synthase, the first pathwayspecific enzyme of pyrrolidine alkaloid biosynthesis, evolved from deoxyhypusine synthase. Proc. Nat. Acad.Sci. 1999, 96, 14777. [7] J. Wainwright, M.M. Schonland. Toxic hepatitis in black patients in Natal. South AfricanMed. J. 1977, 51, 571. [8] R.J. Huxtable, R.A. Cooper. inNatural and Selected Synthetic Toxins: Biological Implications, (Eds:A.T. Tu,W. Gaffield ), American Chemical Society, Washington D.C. 2000, pp: 100–117. [9] F.C Willmot, G.W. Robertson. Senecio disease or cirrhosis of the liver due to Senecio poisoning. Lancet, 1920, 2, 848.
[10] K.A. Beales, K. Betteridge, S.M. Colegate,J.A. Edgar. Solid-phase extraction and LC-MS analysis of pyrrolizidine alkaloids in honeys. J.Agricul. Food Chem., 2004, 52, 6664. [11] K. Betteridge, Y. Cao, S.M. Colegate. Improved method for extraction and LC-MS analysis of pyrrolizidine alkaloids and their N-oxides in honey: Application to Echiumvulgare honeys. J. Agricul. Food Chem., 2005, 53, 1894. [12] A. Dübecke, G. Beckh, C. Lüllmann. Pyrrolizidine Alkaloids in honey and pollen. Food Additives and Contaminants, 2011, 28, 348. [13] M. Kempf, S. Heil, I. Hasslauer, L. Schmidt, K. von der Ohe, C. Theuring, A. Reinhard, P. Schreier, T. Beuerle. Pyrrolizidine alkaloids in pollen and pollen products. Mol. Nut. FoodChem., 2010, 54, 292. [14] J.A. Edgar, L.W. Smith. in Natural and Selected Synthetic Toxins: Biological Implications.Transfer of pyrrolizidine alkaloids into eggs: Food safety implications. (Eds: A.T. Tu, W. Gaffield) ACS Symposium Series, 1999, 745, 118. [15] E. Röder, Medicinal plants in Europe containing pyrrolizidine alkaloids, Pharmazie, 1995, 50, 83. [16] Y-C. Yang, J. Yan, D. R. Doerge, P-C. Chan, P. P. Fu, M. W. Chou. Metabolic Activation of the Tumorigenic Pyrrolizidine Alkaloid, Riddelliine, Leading to DNA Adduct Formation in Vivo.Chem. Res. Toxicol., 2001, 14, 101. [17] E. Roeder. Medicinal plants in China containing pyrrolizidine alkaloids. Pharmazie, 2000, 55, 711. [18] C.D. Dodson, F.R. Stermitz. Pyrrolizidine Alkaloids from Borage (Borago officinalis) Seeds and Flowers. J. Nat. Prod., 1986, 49, 727. [19] K.M. Larson, M.R. Roby,F.R. Stermitz. Unsatured pyrrolizidines from borage (Borago officinalis L.), a common garden herb. J. Nat. Prod., 1984, 47, 747. [20] A.R. Mattocks. Chemistry and toxicology of pyrrolizidine alkaloids, Academic Press, New York, 1986, pp 1–39. [21] N.A. Beauboire, J.E. Simon. Production potential of borage (Borago officinalis L.).Acta Hort., 1987, 208, 101. [22] J.A. Duke. In Handbook of phytochemical constituents of GRAS herbs and other economical plants. CRC Press, London,1992, p 412 [23] I. Wretensiö, L. Svensson, W.W. Christie. Gas-Chromatography- mass spectrometry Identification of the fatty acids in Borage oil using the picolinyl ester derivatives J. Chromatogr.1990, 521, 89. [24] H-J. Mierendorff.Bestimmung von pirrolizidinalkaloidendurchDünn schichtchromatografie in Samenölen von Borago officinalis L.Fta Sci. Technol. 1995, 97, 33. [25] O. Parvais, B. Vander Stricht, R. Vanhaelen-Fastre, M. Vanhaelen. TLC detection of pyrrolizidine alkaloids in oil extracted from the seeds of Borago officinalis. J. Planar Chromatogr.1994, 7, 80. [26] F. Zhang, C-H. Wang, W. Wang. L-X Chen, H-Y Ma, C-F Zhang, M. 2 Zhang, S.W.A. Bligh, Z-T Wang. Quantitative analysis by HPLC-MS of the pyrrolizidine alkaloid adonifoline in Senecio scadens. Phytochem. Anal2008, 19, 25.
1082 wileyonlinelibrary.com/journal/jms
Copyright © 2013 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2013, 48, 1078–1082
Revised: 27 June 2013
Accepted: 5 July 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3251
A rapid and highly specific method to evaluate the presence of pyrrolizidine alkaloids in Borago officinalis seed oil Giulio Vacillotto,a Donata Favretto,b Roberta Seraglia,a* Rita Pagiotti,c Pietro Traldia and Luisa Mattolid Pyrrolizidine alkaloids (PAs) are complex molecules, present in plants as free bases and N-oxides. They are known for their hepatotoxicity, and consequently there is a health risk associated with the use of medicinal herbs that contain PAs. Unfortunately, there is no international regulation of PAs in foods, unlike those for herbs and medicines: in particular, for herbal preparation or herbal extracts, the total PA content must not exceed 1 μg/kg or 1 μg/l, respectively. Borago officinalis seed oil is a source of γ-linolenic acid, and its use is increased in both pharmaceutical and health food industries. Even if studies based on gas chromatography and TLC methods showed that PAs are not co-extracted with oil, the development of a rapid and sensitive method able to evaluate the presence of PAs in commercially available products is surely of interest. The presence of PAs in a commercially available Borago officinalis seed oil was tested either in the oil sample diluted with tetrahydrofuran/methanol (MeOH)/H2O (85/10/5 v:v:v) or after extraction with MeOH/H2O (50/50 v:v) solution The samples were analysed by electrospray ionization in positive ion mode and in high mass resolution (60 000) conditions. In both cases to evaluate the effectiveness of the method, spiking experiments were performed adding known amount of two PA standards to the borage seed oil. A limit of detection in the order of 200 ppt was determined for these two compounds, strongly analogous to Borago officinalis seed oil PAs. Consequently, if present, PAs level in Borago officinalis seed oil must lower than 200 ppt. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Pyrrolizidine alkaloids; Borago officinalis seed oil; high resolution mass spectrometry; tandem mass spectrometry; electrospray ionization
Introduction
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Pyrrolizidine alkaloids (PAs) are complex molecules, consisting of two fused five-membered rings jointed by a single nitrogen atom at position 4 to form a heterocyclic nucleus (see Fig. 1). PAs are plant products produced as secondary metabolites[1] and are sometimes toxic to animals when eaten.[2] Their biological role in plants is quite unknown, but it has been supposed that PAs have evolved as a defense mechanism against unadapted generalist herbivores,[3] while specialist herbivores use this class of compounds for recognizing their food plant, acting as an oviposition stimulant[4] and as feeding stimulant.[5] PAs are present in plants as free bases and as N-oxides (PANOs). More than 600 different structure of PAs and PANOs were identified in over 6000 plant species,[3] and they are mainly present in the families Boraginaceae (all plants), Asteraceae (tribes Senecionaceaea and Eupotoriceae), Orchidaceaea (nine genera) and Fabaceae (mainly the genus Crotalaria).[6] PAs are metabolized in the liver, producing toxic metabolites related to their structure and to the presence of species-specific liver enzymes.[7,8] This toxicity is considered responsible for veno-occlusive liver disease,[9] consisting in the obstruction of the small veins bringing blood from the liver back to the heart. Aside from ingesting the plant directly, PAs may be assumed by eating honey[10–13] and eggs[14] and by drinking milk,[15] produced by animals which have consumed PAs-containing plants. Of concern is the health risk associated with the use of
J. Mass Spectrom. 2013, 48, 1078–1082
medicinal herbs that contain PAs, notably borage leaf, comfrey and colt’s foot in the West, and some Chinese medicinal herbs.[16,17] The European Food Safety Authority was asked by the European Commission to deliver a scientific opinion on PAs in food and feed. The Panel on Contaminants in the Food Chain (CONTAM Panel) performed estimates of both acute and chronic exposure to PAs through honey for three different age groups. Although there might be other sources of PAs exposure, due to lack of data, the CONTAM Panel was not able to quantify dietary exposure from food other than honey. Based on the present knowledge, the CONTAM Panel concluded that 1,2-unsaturated PAs may act as genotoxic carcinogens in humans and supports ongoing efforts to collect more data on the PAs identified and potentially found in feed and relevant foods, including milk, eggs
* Correspondence to: Roberta Seraglia, CNR-ISTM, Corso Stati Uniti 4, 35127 Padova, Italy. E-mail: [email protected] a CNR-ISTM, Corso Stati Uniti 4, I35127 Padova Italy b Department of Molecular Medicine, University Hospital of Padova, I-35121 Padova Italy c Dipartimento di Biologia Vegetale Applicata, Università degli Studi di Perugia Perugia, Italy d Research Area, Aboca S.p.A. Società Agricola, loc. Aboca, Sansepolcro AR, Italy
Copyright © 2013 John Wiley & Sons, Ltd.
Method for evaluating PAs in Borago seed oil
Figure 1. General chemical structure of pyrrolizidine alkaloids and of Borago officinalis pyrrolizidine alkaloids.
J. Mass Spectrom. 2013, 48, 1078–1082
Experimentals Chemicals Tetrahydrofuran (THF), methanol (MeOH), chloroform and water were all HPLC grade and were purchased from Fluka (Milan, Italy).
Samples Borago officinalis oil was supplied by Aboca S.p.A.(Sansepolcro, Italy). Monocrotaline and Retrosine standards were purchased from Sigma–Aldrich (Milan, Italy).
Preparation of Borago officinalis oil sample for analysis. a) Dilution. Aliquots of Borago officinalis oil were diluted in THF/ MeOH/H2O (85:10:5 v:v:v) in ratios ranging from 1: 103 to 1: 106 w/w and vortex mixed for 60 s. In these conditions, a clear solution is obtained, without any phase separation. It is to note that the PAs concentration is necessarily reduced. b) Extraction. 1 ml of MeOH/H2O (50/50 v/v) was added to 250 μl of Borago officinalis oil in a conic, glass vial. After 60 s vortex mixing, the oil was extracted by inversion for 20 min and centrifuged at 13 000 rpm for 10 min. 900 μl of the clear supernatant, containing the PAs fraction, was transferred, and 100 μl of 1% formic acid was added.
Spiking experiments Due to the lack of commercially available reference standards of compounds 1–5, the alkaloids monocrotaline (6) and retrorsine (7) were used as model for the analytical procedures. They were dissolved in MeOH/H2O (50/50 v/v) at concentrations of 32.5 pg/ml, 325 pg/ml, 3.25 ng/ml, 32.5 ng/ml and 325 ng/ml. 100 μl of these solutions was added to a borage seed oil solution in THF/MeOH/H2O (85:10:5 v:v:v) containing 0.1 mg of oil. The solutions so obtained were directly infused into the mass spectrometer. Alternatively, compounds 6 and 7 were dissolved in CHCl3 at concentrations of 200 pg/ml, 2 ng/ml, 20ng/ml, 200ng/ml, 2 μg/ml and 20 μg/ml. 100 μl of these solutions was added to 1 g aliquots of borage oil. The oil aliquots were subsequently extracted as described above with MeOH/H2O.
Copyright © 2013 John Wiley & Sons, Ltd.
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and meat as well as in herbal dietary supplements prepared from PAs-containing plants. PAs are also found in the plant Borago officinalis[18–20](borage), which is an annual herbaceous plant native to Europe, North Africa and Asia Minor.[21] Most of borage PAs have a 1,2-unsaturation in the ring structure indicating hepatotoxicity[20] like amabiline[22] (1), lycopsamine[19] (2), intermidine[19](3) and the acetyl derivatives of lycopsamine and intermidine[19] (4,5) (Fig. 1). During the last ten years, the use of borage seed oil as a source of γ-linolenic acid (GLA) was increased in both pharmaceutical and health food industries. In fact, the oil extracted from the seed of Borago officinalis contains about 20% (w/w) of GLA.[23] Studies carried out by gas chromatography (GC) and TLC methods[18,24,25] showed that PAs are not co-extracted with the oil. These analytical methods can measure the PAs levels in the ppm range for GC[18,24] and in the ppb range for TLC[25] approaches. However, for using borage seed oil in pharmaceuticals, its PAs content must be determined to ensure the complete absence of these compounds. Consequently the development of a rapid and sensitive analytical procedure able to evaluate the presence of PAs in commercially available products is surely of interest. For the analysis of compounds at trace level ( 60 000 (FWHM as defined at m/z 400) and a scan time of 0.65 s. Mass calibration was performed according to the manufacturer’s guidelines using a manufacturer-defined mixture of sodium dodecyl sulphate, sodium taurocholate, the tetrapeptide MRFA (L-methionylarginyl-phenylalanyl-alanine acetate) and Ultramark 1621.
For the investigation on the most relevant alkaloids possibly present in Borago officinalis oil, the direct infusion of diluted oil samples has been employed. To this purpose, the oil was diluted in THF/MeOH/H2O (85:10:5 v:v:v) in the range 1:103- 1:106 and directly infused into the ESI source of the Orbitrap, working in full scan HRMS mode. The best results in terms of signal intensity and stability have been obtained with a dilution 1:104 and with a flow rate of 10 μl/min. As expected, it is particularly complex, and, considering that the ESI conditions employed are not suitable for activating any ‘in source’ fragmentation, it can be considered a fingerprint of the protonated molecular species present in the Borago officinalis oil. In particular, the highly complex ion clusters in the m/z range 800 – 1000 can be ascribed to Na+ adducts of triglycerides present in the oil. However, the direct injection of diluted oil sample does not give any information on the possible presence of the alkaloids typical of Borago officinalis. No peaks corresponding to the [M+H]+ ions of Amabiline (1, accurate mass: 283.1784), lycopsamine (2) and intermidine (3) (accurate mass: 299.1598) and the acetyl derivatives of licopsamine (4) and intermidine (5) (accurate mass: 341.1833) at m/z 284.1856, 300.1676 and 342.1911, respectively, were detected. These results could be due either to the real absence of the alkaloids or to the 1: 104 dilution step prior to analysis that would hamper the detection of components present in the oil at a very low concentration (e.g. at ppb level). In order to verify the latter hypothesis, the oil was extracted with MeOH/H2O (1:1 v:v) to obtain the hydrophilic fraction of the oil in which the PAs should be selectively extracted. Following the preparation reported in the experimental section, the extract so obtained was directly injected in the ESI source. The related spectrum shows again the complete lack in the ESI spectrum of the PAs-related peaks and the disappearance of the ions due the lipophilic species detected in THF/MeOH/H2O.
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Figure 3. Positive ion HRMS spectrum of Borago officinalis seed oil in THF/MeOH/H2O (85/10/5 v/v/v) spiked with 325 ppb of compounds 6 and 7.
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Method for evaluating PAs in Borago seed oil
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Figure 6. MS/MS spectra of [M+H] ions of compounds 6 (a) and 7 (b), added at a concentration of 200 ppt to Borago officinalis seed oil extracted with MeOH/H2O (50/50 v/v).
Figure 4. Plots of concentration of compounds 6 and 7 versus absolute + abundance of their [M+H] ions (m/z 326.1604 for compound 6 and m/z 352.1760 for compound 7) obtained for: (a) dilution with THF/MeOH/H2O (85/10/5 v/v/v) and (b) extraction with MeOH/H2O (50/50 v/v).
+
J. Mass Spectrom. 2013, 48, 1078–1082
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Figure 5. MS/MS spectra of [M+H] ions of compounds 6 (a) and 7 (b), added at a concentration of 32.5 ppb to Borago officinalis seed oil extracted with THF/MeOH/H2O (85/10/5 v/v/v).
Considering that compounds 1–5 are not commercially available, further experiments were carried out by spiking the oil with known, equimolar amounts of two standard alkaloids, respectively monocrotaline (6, MW 325.1511) and retrorsine (7, MW 351.1666). They both lead, in ESI conditions, to abundant [M+H]+ species at m/z 326.1589 and 352.1744, respectively. In order to increase the specificity of the measurements, their behaviour in collisional conditions was studied. The product ion spectra of [M+H]+ of the two alkaloids are reported in Fig. 2, in which the accurate mass values of the fragment ions are reported together with the chemical structures of monocrotaline (Fig. 2a) and retrorsine (Fig. 2b). The first spiking experiment has been performed by adding the standard solutions in the oil diluted in THF/MeOH/H2O, so to obtain standard concentrations in the range 32.5 ppb – 325 ppm. To evaluate the limit of detection (LOD) by direct infusion of the mixture in the ESI source, the presence of [M+H]+of 6 and 7 in high resolution, full scan mode, with a signal-to-noise ratio ≥ 3, was monitored. Even though the spectra are highly complex (see Fig. 3), the two diagnostic peaks are well detectable down to 325 ppb, that can be considered the LOD, as shown in the plot of Fig. 4a, related to the lower concentration used (32.5 ppm – 32.5 ppb). Alternatively to HRMS in full scan mode, an MS/MS approach was also undertook by using the LTQ portion of the Orbitrap. The results obtained are strictly analogous to those achieved in high resolution conditions, with an LOD value of 325 ppb. For the lowest standard concentration (32.5 ppb), the MS/MS spectrum becomes not specific, due to the presence of fragment ions originated from ionic species isobaric with the [M+H]+ ions of the two standard alkaloids (Fig. 5). Considering that a putative concentration of alkaloids in oil at 1 ppm level would become 100 ppt when the oil sample is diluted 1:104 in THF/MeOH/H2O prior to direct infusion, the LODs verified on standards (325 ppb) would not allow the identification of pyrrolyzidine alkaloids at such a low level in borage oil.
G. Vacillotto et al. In the second spiking experiment, the two standard alkaloids 6 and 7 have been added to the oil in a concentration range from 2 ppm to 20 ppt and extracted by MeOH/H2O. By direct analysis of the samples after extraction, in HRMS full scan mode, an LOD in the order of 200 ppt was obtained, as shown in the plot of Fig. 4b, related to the lower concentration used (100 ppb – 20 ppt). Similar LODs were also achieved by using the MS/MS scan approach on the MeOH/water extract, obtaining characteristic fragment products for the [M+H]+ of the two analytes spiked at 200 ppt (Fig. 6). The above described results, though obtained on different alkaloids of the pyrrolizidine family, reasonably suggest that amabiline, lycopsamine, intermidine and the acetyl derivatives of lycopsamine and intermidine, if present in the Borago officinalis oil under investigation, are at level lower than 200 ppt. This rapid, non-chromatographic approach turned out to be valuable for quality control of borage oil preparation. An extraction/enrichment step in the preparation of oil samples was demonstrated to be necessary to reach low LODs at the ppt level.
REFERENCES [1] T. Hartmann, C. Theuring, T. Beuerle, L. Ernst, M.S. Singer, E.A. Bernays. Acquired and partially de novo synthesized pyrrolidine alkaloids in two polyphagusarctiis and the alkaloid profiles of their larval food-plant. J. Chem. Ecol. 2004, 30, 229. [2] D.M. Holstege, J.N. Seiber, F.D. Galey. Rapid multiresidue screen for alkaloids in plant material and biological sample. J. Agricul. Food Chem. 1995, 43, 691. [3] L.J. Doorduin, K. Vrieling. A review of the phytochemical support for shifting defence hypothesis. Phytochem. Rev. 2011, 10, 99. [4] M. Màčel, K. Vrielin. Pyrrolizidine alkaloids as oviposition stimulants for the cinnabar moth, Tyriajacobaeae. J. Chem. Ecol. 2003, 29, 1435. [5] E.A. Bernays, T. Hartmann, R.F. Chapman. Gustatory responsiveness to pyrrolizidine alkaloids in the Senecio specialist, Tyriajacobaeae (Lepidoptera, Actiidae). Phys. Entomol. 2004, 29, 67. [6] D. Oder, T. Hartmann. Homospermidine synthase, the first pathwayspecific enzyme of pyrrolidine alkaloid biosynthesis, evolved from deoxyhypusine synthase. Proc. Nat. Acad.Sci. 1999, 96, 14777. [7] J. Wainwright, M.M. Schonland. Toxic hepatitis in black patients in Natal. South AfricanMed. J. 1977, 51, 571. [8] R.J. Huxtable, R.A. Cooper. inNatural and Selected Synthetic Toxins: Biological Implications, (Eds:A.T. Tu,W. Gaffield ), American Chemical Society, Washington D.C. 2000, pp: 100–117. [9] F.C Willmot, G.W. Robertson. Senecio disease or cirrhosis of the liver due to Senecio poisoning. Lancet, 1920, 2, 848.
[10] K.A. Beales, K. Betteridge, S.M. Colegate,J.A. Edgar. Solid-phase extraction and LC-MS analysis of pyrrolizidine alkaloids in honeys. J.Agricul. Food Chem., 2004, 52, 6664. [11] K. Betteridge, Y. Cao, S.M. Colegate. Improved method for extraction and LC-MS analysis of pyrrolizidine alkaloids and their N-oxides in honey: Application to Echiumvulgare honeys. J. Agricul. Food Chem., 2005, 53, 1894. [12] A. Dübecke, G. Beckh, C. Lüllmann. Pyrrolizidine Alkaloids in honey and pollen. Food Additives and Contaminants, 2011, 28, 348. [13] M. Kempf, S. Heil, I. Hasslauer, L. Schmidt, K. von der Ohe, C. Theuring, A. Reinhard, P. Schreier, T. Beuerle. Pyrrolizidine alkaloids in pollen and pollen products. Mol. Nut. FoodChem., 2010, 54, 292. [14] J.A. Edgar, L.W. Smith. in Natural and Selected Synthetic Toxins: Biological Implications.Transfer of pyrrolizidine alkaloids into eggs: Food safety implications. (Eds: A.T. Tu, W. Gaffield) ACS Symposium Series, 1999, 745, 118. [15] E. Röder, Medicinal plants in Europe containing pyrrolizidine alkaloids, Pharmazie, 1995, 50, 83. [16] Y-C. Yang, J. Yan, D. R. Doerge, P-C. Chan, P. P. Fu, M. W. Chou. Metabolic Activation of the Tumorigenic Pyrrolizidine Alkaloid, Riddelliine, Leading to DNA Adduct Formation in Vivo.Chem. Res. Toxicol., 2001, 14, 101. [17] E. Roeder. Medicinal plants in China containing pyrrolizidine alkaloids. Pharmazie, 2000, 55, 711. [18] C.D. Dodson, F.R. Stermitz. Pyrrolizidine Alkaloids from Borage (Borago officinalis) Seeds and Flowers. J. Nat. Prod., 1986, 49, 727. [19] K.M. Larson, M.R. Roby,F.R. Stermitz. Unsatured pyrrolizidines from borage (Borago officinalis L.), a common garden herb. J. Nat. Prod., 1984, 47, 747. [20] A.R. Mattocks. Chemistry and toxicology of pyrrolizidine alkaloids, Academic Press, New York, 1986, pp 1–39. [21] N.A. Beauboire, J.E. Simon. Production potential of borage (Borago officinalis L.).Acta Hort., 1987, 208, 101. [22] J.A. Duke. In Handbook of phytochemical constituents of GRAS herbs and other economical plants. CRC Press, London,1992, p 412 [23] I. Wretensiö, L. Svensson, W.W. Christie. Gas-Chromatography- mass spectrometry Identification of the fatty acids in Borage oil using the picolinyl ester derivatives J. Chromatogr.1990, 521, 89. [24] H-J. Mierendorff.Bestimmung von pirrolizidinalkaloidendurchDünn schichtchromatografie in Samenölen von Borago officinalis L.Fta Sci. Technol. 1995, 97, 33. [25] O. Parvais, B. Vander Stricht, R. Vanhaelen-Fastre, M. Vanhaelen. TLC detection of pyrrolizidine alkaloids in oil extracted from the seeds of Borago officinalis. J. Planar Chromatogr.1994, 7, 80. [26] F. Zhang, C-H. Wang, W. Wang. L-X Chen, H-Y Ma, C-F Zhang, M. 2 Zhang, S.W.A. Bligh, Z-T Wang. Quantitative analysis by HPLC-MS of the pyrrolizidine alkaloid adonifoline in Senecio scadens. Phytochem. Anal2008, 19, 25.
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