Survey of pyrrolizidine alkaloids in seven varieties of Lappula squarrosa: An alternative source of heart-healthy vegetable oil Emmanuel Letsyo, a Gerold Jerz, a Peter Winterhalter, a Gert Horn b and Till Beuerle c * ABSTRACT: Introduction – Growing demand for heart-healthy omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is putting stress on wild fish stocks. There is now a compelling need for new and novel sources of non-traditional seed oils containing high stearidonic acid (SDA), a precursor of EPA and DHA, to reduce this demand. The seed oil of Lappula squarrosa is one of the richest sources of SDA, however, the plant has been found to contain toxic pyrrolizidine alkaloids (PAs). Objective – In this study, the PA concentrations of seven varieties (A–G) of Lappula squarrosa were analysed to determine the most suitable varieties for commercial seed oil production. Methods – Whilst the clean-up procedure for the PAs in the roots, flowers and leaves was on diatomaceous earth columns and finally analysed with GC-EI-MS, that of the seeds was through SCX-SPE and a more sensitive HPLC-ESI-MS/MS sum parameter method was used in the analysis. Results – Altogether six PAs (supinine, amabiline, intermedine, lycopsamine and 3ʹ-acetylintermedine) including one unknown retronecine-type PA were identified with variety C recording the lowest total PA concentration (4.64 mg seneciphylline equiva- lents (SE)/g dry weight (d.w.)). Besides, the total PA concentrations in the seeds of Lappula squarrosa varieties ranged between 2.88 μg PA/g and 10.36 μg PA/g d.w. Conclusion – Based solely on overall PA concentrations and PA distribution, variety D (5.95 mg SE/g d.w.) was found to be a potential candidate for commercial seed oil cultivation. Copyright © 2016 John Wiley & Sons, Ltd. Supporting information can be found in the online version of this article. Keywords: Lappula squarrosa; pyrrolizidine alkaloid; Boraginaceae; non-traditional seed oil; GC-EI-MS; HPLC-ESI-MS/MS; sum parameter; food safety Introduction In recent years, there has been immense interest in health- beneficial seed oils due to reports that suggest that it could be used for the production of vegetable oils with unique cardiovascu- lar benefits. Besides, over-fishing of the seas, increasing world pop- ulation and generally low amounts of omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) in major oil crops have all fuelled the debate on the possibility of using an alternative or non-traditional seed crop for heart-healthy vegetable oils. This de- bate has been escalated due to a recent report that demonstrates that heart diseases are the leading cause of death among 15 major causes of death in the United States over the past five decades (Kochanek et al., 2011). Fish, shellfish and algal oils are the main sources of these health-beneficial n-3 LC-PUFA, eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3), however, over-fishing of the seas is leading to dwindling fortunes of these products. Consequently, there is now a pressing need for new and novel sources of heart-healthy long-chain omega-3 fatty acids to help consumers meet their daily dietary requirements. Lappula squarrosa (Retz.) Dumort (European stickseed) belongs to the Boraginaceae family and are introduced species in North America and Africa, even though, native to Europe and Asia (Agro atlas: Interactive Agricultural Ecological Atlas of Russia and Neighboring Countries, 2009). The plant is a summer or winter annual herb but biennial forms are common as well. Lappula squarrosa usually grows along the roadsides, waste areas and cul- tivated fields, and each plant is capable of producing between 200 and 40000 seeds depending on the season (Frick, 1984; Royer and Dickinson, 1999). However, typical seed production of the culti- vated plants investigated in this study ranged between 700 and 6000 per plant (Exsemine, 2010). The seed oil of L. squarrosa is one of the richest sources of PUFA known within the Boraginaceae family. In fact, the fatty acids, especially n-3 LC-PUFA, are believed to play an essential role in the growth and development of the brain, the regulation of blood pressure, renal function, blood * Correspondence to: Till Beuerle, Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Mendelssohnstrasse 1, 38106 Braun- schweig, Germany. E-mail: t.beuerle@tu-braunschweig.de a Institut für Lebensmittelchemie, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany b Exsemine GmbH, Am Wehr 4, 06198 Salzatal, Germany c Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Mendelssohnstrasse 1, 38106 Braunschweig, Germany Phytochem. Anal. 2016, 27, 133–139 Copyright © 2016 John Wiley & Sons, Ltd. Research article Received: 20 October 2015, Revised: 27 November 2015, Accepted: 5 December 2015 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/pca.2608 133