RESEARCH PAPER Analytical strategy for determination of known and unknown destruxins using hybrid quadrupole-Orbitrap high-resolution mass spectrometry Natalia Arroyo-Manzanares 1,2 & José Diana Di Mavungu 1 & Inmaculada Garrido-Jurado 3 & Lourdes Arce 2 & Lynn Vanhaecke 4 & Enrique Quesada-Moraga 3 & Sarah De Saeger 1 Received: 22 December 2016 /Revised: 16 February 2017 /Accepted: 22 February 2017 /Published online: 9 March 2017 # Springer-Verlag Berlin Heidelberg 2017 Abstract An analytical strategy based on a hybrid quadrupole- Orbitrap mass spectrometry was proposed for the simultaneous screening of known destruxins and characterization of potential members of this class of secondary metabolites, in order to evaluate the metabolite production of entomopathogenic fungi used as biocontrol agents. Initially, the fragmentation pathway of the known and commercially available destruxin A was established combining high resolution mass spectrometry (HRMS) and multiple stage MS data in order to obtain the strategy for the characterization of other destruxins for which reference standards were not available. Nineteen known destruxins including A, B, C, D, Ed, F, A 1 ,B 1 , Ed 1 ,A 2 ,B 2 , D 2 ,A 3 , DesmA, DesmB, DesmC, DesmB 2 , and two chloro- derivatives (Cl and E 2 chlorohydrin) were unequivocally iden- tified in Metarhizium brunneum using the proposed strategy. In addition, four unknown destruxins, namely C 1 , Ed 2 , G, and G 1 , were structurally elucidated and characterized for the first time in this fungal strain. Keywords Destruxins identification . Untargeted analysis . Hybrid quadrupole-Orbitrap . Fragmentation pathway Introduction Destruxins are fungal secondary metabolites produced mainly by entomopathogenic fungus Metarhizium spp. such as Metarhizium anisopliae [1, 2] or Metarhizium brunneum [3], as well as by other fungi including Aschersonia aleyrodis [4], Alternaria brassicae [5], Trichotecium roseum [6], Beauveria felina [7], Nigrosabulum globosum [8], and imperfect fungus [9]. Destruxins are cyclic hexadepsipeptides composed of an α- hydroxy acid and five amino acid (AA) residues (Fig. 1). The configurations of the amino acid residues are S form, and those of the hydroxy acids are R form. Since the first isolation of destruxins A and B from M. anisopliae [1], 40 different destruxins were identified in several fungal strains. They can be classified into seven subgroups named A, B, C, D, E, E-diol (Ed), and F depending on the hydroxy acid group. Moreover, different subscripts are assigned depending on the different constituent amino acids. Thereby, subscript 1 (X 1 ) indicates pipecolic acid instead of proline residue at position 2, while the replacement of isoleucine by valine at position 3 is desig- nated with subscript 2 (X 2 ). Subscript 3 (X 3 ), 4 (X 4 ), and 5 (X 5 ) have been only used for A series. Subscript 3 designates the Natalia Arroyo-Manzanares and José Diana Di Mavungu contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0276-z) contains supplementary material, which is available to authorized users. * Natalia Arroyo-Manzanares qa2arman@uco.es * José Diana Di Mavungu Jose.DianaDiMavungu@UGhent.be 1 Faculty of Pharmaceutical Sciences, Laboratory of Food Analysis, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium 2 Department of Analytical Chemistry, University of Cordoba, Annex C3 Building, Campus of Rabanales, 14071 Cordoba, Spain 3 Department of Agricultural and Forestry Sciences, ETSIAM, University of Cordoba, Building C4, Campus of Rabanales, 14071 Cordoba, Spain 4 Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Anal Bioanal Chem (2017) 409:3347–3357 DOI 10.1007/s00216-017-0276-z