ANALYTICAL AND LABORATORY EQUIPMENT 29
Analysis of mycotoxins in grains
Brian Kinsella explores a method to prepare samples using polymeric SPE and LC-MS-MS.
M ycotoxins are
naturally occurring secondary metabolites
produced by several species of fungi. Tey pose a health risk to humans and animals due to their harmful biological properties and common occurrence in food and feed. Analysis of mycotoxins is challenging due to their large number and varied physicochemical properties. Additionally, the food samples tested are complex in nature and are often contaminated with several mycotoxins at low concentrations.
Fig. 1. SRM Transitions.
Tis application details a method for the sample preparation of grain-based food via polymeric SPE and analysis by LC-MS/ MS. HPLC separation of 16 mycotoxins and three internal standards was successfully conducted in < 16 minutes using a Selectra DA column – 100 × 2.1mm, 3 µm from UCT. Tis
polyaromatic column provides greater retention of the polar trichothecenes as compared to a C18 column. Compounds included are representative of a wide range of mycotoxins, including type A- and B-trichothecenes, ochratoxin A, alternariol, zearalenone, α- & β-zearalanol and aflatoxins.
2g of sample was hydrated with 2ml of DI water (≥15 min). 10ml of ACN was added to each sample and vortex mixed for 10 minutes followed by centrifugation for 10 minutes (≥3,000 × g, 4°C). Te supernatant was transferred to a clean glass tube and evaporated to dryness at 50°C under a gentle stream of nitrogen. 10ml of DI water was added to each sample and vortex mixed.
SPE procedure Polymeric SPE cartridges (EnviroClean HL DVB 200 mg/6ml cartridge from UCT)
were conditioned with 3ml MeOH and 3ml DI water. Te sample supernatants were loaded onto each cartridge and allowed to percolate through the cartridges under gravity. Te cartridges were then washed sequentially with 3ml of DI water followed by 3ml of 10% MeOH. Te cartridges were dried under vacuum for 10 minutes (≥10 inHg). Ten 3ml of hexane was added to each cartridge and slowly drawn through. Te cartridges were dried under vacuum (≥10 inHg) for five minutes. Te mycotoxins were eluted from the SPE cartridges using 4ml ACN. Te extracts were evaporated to dryness at 40°C under a gentle stream of nitrogen, then reconstituted in 1ml of MeOH:H2
O (50:50, v/v).
Te instrumentation used for this experiment was a Termo Scientific Dionex Ultimate 3000 LC system coupled to a Termo Scientific TSQ Vantage tandem mass spectrometer. Various LC-MS/MS conditions were examined prior to settling on the use of APCI ionisation and MeOH/ammonium formate (10mm) as the mobile phase. Tis set-up provided the best combination to effectively detect and quantitate all the mycotoxins. Parameters investigated included ESI versus APCI; ACN versus MeOH as the organic solvent, as well as a variety of mobile phase buffers.
Owing to their polarity, type-B trichothecenes typically elute early in chromatographic runs, and are known to be prone to matrix effects in the ion source. To reduce this possibility a Selectra DA column containing a polyaromatic phase was
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