Planta Med 2015; 81(18): 1736-1743
DOI: 10.1055/s-0035-1557823
Analytical Studies
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

Judith Taibon
1   Institute of Pharmacy, Department of Pharmacognosy, CCB – Centrum of Chemistry and Biomedicine, University of Innsbruck, Innsbruck, Austria
2   Institute of Microbiology, University of Innsbruck, Innsbruck, Austria
Sonja Sturm
1   Institute of Pharmacy, Department of Pharmacognosy, CCB – Centrum of Chemistry and Biomedicine, University of Innsbruck, Innsbruck, Austria
Christoph Seger
1   Institute of Pharmacy, Department of Pharmacognosy, CCB – Centrum of Chemistry and Biomedicine, University of Innsbruck, Innsbruck, Austria
3   Institute of Medical and Chemical Laboratory Diagnostics (ZIMCL), University Hospital Innsbruck, Innsbruck, Austria
Marion Werth
1   Institute of Pharmacy, Department of Pharmacognosy, CCB – Centrum of Chemistry and Biomedicine, University of Innsbruck, Innsbruck, Austria
Hermann Strasser
2   Institute of Microbiology, University of Innsbruck, Innsbruck, Austria
Hermann Stuppner
1   Institute of Pharmacy, Department of Pharmacognosy, CCB – Centrum of Chemistry and Biomedicine, University of Innsbruck, Innsbruck, Austria
› Author Affiliations
Further Information

Publication History

received 23 April 2015
revised 01 July 2015

accepted 02 July 2015

Publication Date:
14 September 2015 (online)


A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02 % trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2 % to 20 %, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

  • References

  • 1 Dispas A, Lebrun P, Ziemons E, Marini R, Rozet E, Hubert P. Evaluation of the quantitative performances of supercritical fluid chromatography: from method development to validation. J Chromatogr A 2014; 1353: 78-88
  • 2 Taylor LT. Supercritical fluid chromatography. Anal Chem 2008; 80: 4285-4294
  • 3 Taylor LT. Supercritical fluid chromatography. Anal Chem 2010; 82: 4925-4935
  • 4 Bhatt HS, Patel GF, Vekariya NV, Jadav SK. Super critical fluid chromatography–an overview. J Pharm Res 2009; 2: 1606-1611
  • 5 Dispas A, Lebrun P, Andri B, Rozet E, Hubert P. Robust method optimization strategy–a useful tool for method transfer: the case of SFC. J Pharm Biomed Anal 2014; 88: 519-524
  • 6 Berger TA. Demonstration of high speeds with low pressure drops using 1.8 µm particles in SFC. Chromatographia 2010; 72: 597-602
  • 7 Grand-Guillaume Perrenoud A, Veuthey J, Guillarme D. Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the analysis of pharmaceutical compounds. J Chromatogr A 2012; 1266: 158-167
  • 8 Sarazin C, Thiébaut D, Sassiat P, Vial J. Feasibility of ultra high performance supercritical neat carbon dioxide chromatography at conventional pressures. J Sep Sci 2011; 34: 2773-2778
  • 9 Bischoff JF, Rehner SA, Humber RA. A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 2009; 101: 512-530
  • 10 Kepler RM, Rehner SA. Genome-assisted development of nuclear intergenic sequence markers for entomopathogenic fungi of the Metarhizium anisopliae species complex. Mol Ecol Resour 2013; 13: 210-217
  • 11 Petzold-Maxwell JL, Jaronski ST, Clifton EH, Dunbar MW, Jackson MA, Gassmann AJ. Interactions among Bt maize, entomopathogens, and rootworm species (Coleoptera: Chrysomelidae) in the field: effects on survival, yield, and root injury. J Econ Entomol 2013; 106: 622-632
  • 12 Pedras MSC, Irina Zaharia L, Ward DE. The destruxins: synthesis, biosynthesis, biotransformation, and biological activity. Phytochemistry 2002; 59: 579-596
  • 13 Zimmermann G. Review on safety of the entomopathogenic fungus Metarhizium anisopliae . Biocontrol Sci Technol 2007; 17: 879-920
  • 14 Dornetshuber-Fleiss R, Heffeter P, Mohr T, Hazemi P, Kryeziu K, Seger C, Berger W, Lemmens-Gruber R. Destruxins: fungal-derived cyclohexadepsipeptides with multifaceted anticancer and antiangiogenic activities. Biochem Pharmacol 2013; 86: 361-377
  • 15 Dumas C, Matha V, Quiot JM, Vey A. Effects of destruxins, cyclic depsipeptide mycotoxins, on calcium balance and phosphorylation of intracellular proteins in lepidopteran cell lines. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 1996; 114: 213-219
  • 16 Hinaje M, Ford M, Banting L, Arkle S, Khambay B. An investigation of the ionophoric characteristics of destruxin A. Arch Biochem Biophys 2002; 405: 73-77
  • 17 Nakagawa H, Takami M, Udagawa N, Sawae Y, Suda K, Sasaki T, Takahashi N, Wachi M, Nagai K, Woo JT. Destruxins, cyclodepsipeptides, block the formation of actin rings and prominent clear zones and ruffled borders in osteoclasts. Bone 2003; 33: 443-455
  • 18 Tsunoo A, Kamijo M. Non-cyclic AMP-dependent, positive inotropic cyclodepsipeptides with negative chronotropy. J Pharmacol Exp Ther 1999; 290: 1006-1012
  • 19 Buchwaldt L, Jensen J. HPLC purification of destruxins produced by Alternaria brassicae in culture and leaves of Brassica napus . Phytochemistry 1991; 30: 2311-2316
  • 20 Hsiao YM, Ko JL. Determination of destruxins, cyclic peptide toxins, produced by different strains of Metarhizium anisopliae and their mutants induced by ethyl methane sulfonate and ultraviolet using HPLC method. Toxicon 2001; 39: 837-841
  • 21 Jegorov A, Havlíček V, Sedmera P. Rapid screening of destruxins by liquid chromatography/mass spectrometry. J Mass Spectrom 1998; 33: 274-280
  • 22 Jegorov A, Paizs B, Žabka M, Kuzma M, Havlíček V, Giannakopulos A, Derrick P. Profiling of cyclic hexadepsipeptides roseotoxins synthesized in vitro and in vivo: a combined tandem mass spectrometry and quantum chemical study. Eur J Mass Spectrom 2003; 9: 105-116
  • 23 Kershaw MJ, Moorhouse ER, Bateman R, Reynolds SE, Charnley AK. The role of destruxins in the pathogenicity of Metarhizium anisopliae for three species of insect. J Invertebr Pathol 1999; 74: 213-223
  • 24 Loutelier C, Cherton J, Lange C, Traris M, Vey A. Studies on the dynamics of the production of destruxins by Metarhizium anisopliae direct high-performance liquid chromatographic and fast atom bombardment mass spectrometric analysis correlated with biological activity tests. J Chromatogr A 1996; 738: 181-189
  • 25 Morais RP, Lira SP, Seleghim MHR, Berlinck RGS. A method for dextruxin analysis by HPLC-PDA-ELSD-MS. J Braz Chem Soc 2010; 21: 2262-2271
  • 26 Potterat O, Wagner K, Haag H. Liquid chromatography-electrospray time-of-flight mass spectrometry for on-line accurate mass determination and identification of cyclodepsipeptides in a crude extract of the fungus Metarrhizium anisopliae . J Chromatogr A 2000; 872: 85-90
  • 27 Seger C, Sturm S, Stuppner H, Butt TM, Strasser H. Combination of a new sample preparation strategy with an accelerated high-performance liquid chromatography assay with photodiode array and mass spectrometric detection for the determination of destruxins from Metarhizium anisopliae culture broth. J Chromatogr A 2004; 1061: 35-43
  • 28 Wang C, Skrobek A, Butt TM. Concurrence of losing a chromosome and the ability to produce destruxins in a mutant of Metarhizium anisopliae . FEMS Microbiol Lett 2003; 226: 373-378
  • 29 Wang C, Skrobek A, Butt TM. Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae . J Invertebr Pathol 2004; 85: 168-174
  • 30 Butt TM, Hadj NBE, Skrobek A, Ravensberg WJ, Wang C, Lange CM, Vey A, Shah UK, Dudley E. Mass spectrometry as a tool for the selective profiling of destruxins; their first identification in Lecanicillium longisporum . Rapid Commun Mass Spectrom 2009; 23: 1426-1434
  • 31 Taibon J, Sturm S, Seger C, Parth M, Strasser H, Stuppner H. Development of a fast and selective UHPLC-DAD-QTOF-MS/MS method for the qualitative and quantitative assessment of destruxin profiles. Anal Bioanal Chem 2014; 406: 7623-7632
  • 32 Liu C, Huang S, Tzeng Y. Analysis of destruxins produced from Metarhizium anisopliae by capillary electrophoresis. J Chromatogr Sci 2004; 42: 140-144
  • 33 Taylor LT. Packed column supercritical fluid chromatography of hydrophilic analytes via water-rich modifiers. J Chromatogr A 2012; 1250: 196-204
  • 34 Seger C, Eberhart K, Sturm S, Strasser H, Stuppner H. Apolar chromatography on Sephadex LH-20 combined with high-speed counter-current chromatography. J Chromatogr A 2006; 1117: 67-73
  • 35 ICH Harmonized Tripartite Guideline. Validation of analytical procedures: text and methodology Q2(R1). Available at Accessed April 23, 2015