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

A method is presented allowing the qualitative and quantitative analysis of destruxins (dtxs) in fungal culture broth. Sample preparation was carried out by ultrafiltration over a commercially available acetylated cellulose (CTA) membrane with a Mr 10000 cut-off. The developed high-performance liquid chromatography assay with diode array detection (HPLC–DAD) cuts down the analysis time by 50% compared to most of the currently applied methods (retention times: dtx A = 8.3 min, dtx B = 8.9 min, dtx E = 7.5 min) and enables dtx detection down to sub-ppm range (limits of detection: dtx A = 0.19 mg/l, dtx B = 0.41 mg/l, dtx E = 0.10 mg/l). Stability of dtx E in filtrated culture broth was found to be much lower than anticipated (half-life time = 64.5 ± 1.7 h). Thus, the detoxification of this metabolite is an abiotic process. Coupling of the HPLC–DAD system to an ion trap mass spectrometer with an electrospray ionization source operating in the positive mode allowed identification of most dtxs encountered by utilizing multiple stage MS–MS experiments and retention time rules.