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.
Seger
Christoph
C
Institute of Pharmacy, Leopold Franzens University Innsbruck, Innrain 52, A-6020 Innsbruck, Austria. christoph.seger@uibk.ac.at
Sturm
Sonja
S
Stuppner
Hermann
H
Butt
Tariq M
TM
Strasser
Hermann
H
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003470:Culture Media; D047630:Depsipeptides / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D003470:Culture Media; D047630:Depsipeptides