SCREENING FR FUNGAL AND BACTERIAL METABLIES USING QTRAP TECNLGY Michael Sulyok Center for Analytical Chemistry Department IFA-Tulln University of Natural Resources and Life Sciences Vienna Analytica 2016 Munich, May 11 th, 2016
Definition Mycotoxins Low molecular weight-secondary metabolites produced by molds; toxic for humans and animals Formation Aspergillus sp., Penicillium sp., Alternaria sp. Fusarium sp., Usually synthesis of a group of compounds Function Chemical weapons against hosts and competitors ccurrence 25% of all grains contaminted world-wide (FA) Most important chronic dietary risk-factor Economic impact (1.5 bn $ annual loss for US crop production) Regulatory limits
Nature of Mycotoxins 3 C C 3 C3 3 C C2 C3 Nivalenol (very polar) C 3 C 2 I Ac Ac 3 C T-2 Toxin chratoxin A (non-uv-absorber) (fluorescent) 2 C 3 C Deoxynivalenol (UV-absorber) C N Cl C 3 Major differences: polarities UV absorption and fluorescence ionic nature (dependent on p) occurrence in different commodities concentration levels of interest [C] [C] C 3 C 3 C 3 N 2 Fumonisins (acidic) 3 C C 3 3 C C 3 C 2 I Ac (K) C 3 [C] [C] C 3 T-2 Toxin Moniliformin Zearalenone Aflatoxin B1 (non-uv-absorber) (ionic) (apolar, fluorescent) 3
Analysis scheme Agilent 1290 PLC Extraction (90 min) 5g sample + Phenomenex Gemini C18, 150x4.6 mm, 5µm 20mL ACN+ 2 +Ac 79+20+1 v+v+v 1 ml/min flow rate + Dilution (1+1) with ACN/ 2 /Ac 20/79/1 AB SCIEX QTRAP 5500 in scheduled MRM mode; 2 injections (pos/neg) 710 analytes (May 2016) Intensity, cps 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 3.23 2 4 6 8 Time, 10 min 12 14 16 18 20 Analysis 5 µl of diluted raw extract injected without further pretreatment Matrix effects?! No UPLC?!
Estimated Dwell Time (ms) Reverse Phase Gradient 275 250 225 200 175 150 125 100 75 50 25 0 Estimated Dwell Time for MRM across Time 0 0 5 10 15 20 Time (mins) Excel-Macro provided by Andre Schreiber, Sciex 886 MRMs 1000 ms cycle time 40 s MRM window width More options (individual window widths, dwell time weighting) in the advanced smrm mode 100 90 80 70 60 50 40 30 20 10
Compilaton of z-scores obtained by our method in routine proficiency testing (BIPEA) 4 3 2 1 0-1 -2-3 -4 maize grains nuts baby food animal feed raisins, pepper, coffee, milk.
Case study: Semi-automated production of sprouted barley as feed for cattle Diagnosis: Tremor
Case study: Semi-automated production of sprouted barley as feed for cattle Black spots: Aspergillus sp.?
LC-MS/MS (+) analysis 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 Kojic acid 114 mg/kg Cytochalasin E 7 mg/kg 1.2e6 1.1e6 Intensity, cps 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 0.0 Fusarinolic acid 8 mg/kg Fusaric acid 3.8 mg/kg Bikaverin 20 mg/kg + Fumonisins (2 mg/kg) + Enniatins (200 µg/kg) + Tryptoquivaline derivatives 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 Time, min
LC-MS/MS (+) analysis 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 Kojic acid 114 mg/kg Cytochalasin E 7 mg/kg 1.2e6 1.1e6 Intensity, cps 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 0.0 ESI (-) Fusarinolic acid Patulin 5008 µg/kg mg/kg Pseurotin A 900 µg/kg Patulin, Pseurotin Fusaric A and acid Cytochalasin E confirm A. clavatus 3.8 mg/kg Fusarin C 7.6 mg/kg Bikaverin 20 mg/kg + Fumonisins (2 mg/kg) + Enniatins (200 µg/kg) + Tryptoquivaline derivatives 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 Time, min
The mycotoxin issue in cocoa After freshly harvested pods are opened, beans undergo natural fermentation Toxigenic fungi may contaminate beans mainly during fermentation, but also during processing/storage Main toxigenic species: A. flavus, A. carbonarius, A. fumigatus, A. versicolor, A. niger Mycotoxin data exist for aflatoxins and TA only; usually low-medium prevalence, low concentrations
LC-ESI(+)-MS/MS chromatogram of CG 14 5.5e5 5.0e5 4.5e5 4.0e5 Sterigmatocystin 149 µg/kg 3.5e5 Intensity, cps 3.0e5 2.5e5 2.0e5 No AFB1 or TA! ESI (-): STERA precursors Cyclopiazonic acid 246 µg/kg 1.5e5 1.0e5 5.0e4 0.0 2.99 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 Time, min
Prevalence of selected metabolites Analyte No. of positives Median of pos. (µg/kg) Maximum (µg/kg) Aflatoxin B1 6 / 57 12.9 270 chratoxin A 14 / 57 4.9 11.0 Cyclopiazonic acid 12 / 57 343 4320 Sterigmatocystin 57 / 57 1.86 1910 Averufin 46 / 57 0.89 225 Pseurotin A 39 / 57 123 1890 Nigragillin 57 / 57 n.a. n.a. Nonactin 48 / 57 1.92 613 Metabolite pattern suggests that afla- and ochratoxigenic Aspergilli are less prevalent than A. versicolor, A. fumigatus and A. niger Nonactin indicates presence of Streptomyces sp. verall more than 150 different metabolites
Relevance for the consumer? XIC of -MRM (510 pairs): 367.000/295.100 amu Expected RT: 15.7 ID: Averufin.1 from Sample 1 (AT5-1881-2) of neg15.wiff Max. 7954.0 (Turbo cps. Spray) Very preliminary data indicates that sterigmatocystin may be present in cocoa and related products sold in Austria (4/10 pos) Intensity, cps 7954 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 XIC of STERA in Cocoa Brand B 11.5 µg/kg 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 Time, min 15.62 Contribution of cocoa and related products to the overall exposure to sterigmatocystin is not negligible - 1 / 1259 samples > 11.5 µg/kg (EFSA survey 1 ) - significant consumption by children 2 1 http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/774e.pdf 2 Copetti MV et al., Int J Food Microbiol (2014) 178:13-20
utlook: Multi-class methods 1.3e6 1.2e6 1.1e6 Survey in fufu from Nigeria 1.0e6 9.0e5 8.0e5 Intensity, cps 7.0e5 6.0e5 5.0e5 4.0e5 180 µg/kg cereulide 3.0e5 2.0e5 1.0e5 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 Time, min
Conclusions The LC-MS/MS based dilute-and-shoot approach targeting several hundreds of analytes is sufficiently accurate (acceptable z-scores in proficiency testing) In extreme cases, methods limited to routinemycotoxins fail to come up with the correct answer on the hazards posed by MTs in a particular sample Generation of comprehensive data sets on prevalence of mycotoxins / fungal metabolites feasible Identifiation of unexpected exposure scenarios Co-occurrence of mycotoxins (and other contaminants)