Dr Shun-Hsin Liang & Dr Jamie York explore simultaneous determination of alternaria toxins, ergot alkaloid epimers and other major mycotoxins in various food matrices by LC-MS/MS
As more mycotoxins come under regulatory purview, new methods are needed to help food safety labs continue to operate efficiently. Comprehensive multi-mycotoxin methods are an attractive alternative to separate methods for different analyte lists, but they can be difficult to develop due to the wide range of chemical characteristics among mycotoxin classes. In particular, the alternaria toxins and ergot alkaloids create additional challenges for method developers. These emerging mycotoxins are unique in that, when analysed on a C18 column, high pH conditions must be used to obtain acceptable peak shape for the Alternaria toxins and for adequate separation of the ergot alkaloid epimers. Use of high pH conditions is stressful for LC columns and not suitable for analysing other classes of mycotoxins, so another approach is required for a truly comprehensive method.
Another factor that complicates any comprehensive method is the need to mitigate matrix suppression or enhancement. Due to the variation in mycotoxin chemical characteristics, it is unlikely that a single clean-up protocol would produce proper recoveries and accurate quantification for all types of mycotoxins.
A unique, simplified solution
Simultaneous analysis of 37 mycotoxins was achieved with a simple sample preparation and a fast, 11-minute run time on a Raptor Biphenyl LC column under acidic conditions. Importantly, all epimer pairs of ergot alkaloids were chromatographically separated for definitive and accurate quantification, and quantifiable peak shape was obtained for tenuazonic acid.
Four food matrices (wheat baby cereal, peanut, tomato puree and blended flour) were used to demonstrate the applicability of this method to a wide range of food types. Samples were extracted in an 80:20 acetonitrile:water solution containing 0.5% formic acid (no formic acid was required for tomato puree extraction). After drying, samples were reconstituted in 50:50 water:methanol to produce more symmetrical peaks for early eluting compounds and ergot alkaloids and to increase the overall signal intensity, especially for fumonisin B compounds.
Excellent chromatographic results achieved under acidic conditions
Overall, the combination of a simple, universal sample preparation and analysis on a Raptor Biphenyl column under acidic conditions produced excellent results for most mycotoxins (See Fig.1), demonstrating that the method is suitable for quantitative analysis. The matrix-matched external standard calibration was implemented for quantification. To assess linearity, quadratic regression (1/x weighted) produced the best fit calibration curves for all analytes. Most analytes were quantifiable across the full range of 0.4-400 µg/kg (of sample concentrations), and all compounds showed proper linearity with r2 >0.997 and deviations <30%.
To assess accuracy and precision, three batches were analysed on three different days, giving a total of nine replicates for each fortification level in each commodity. Good recoveries (72-112%) were obtained for nearly all compounds across all fortification levels and matrices, demonstrating acceptable method accuracy. Satisfactory method precision was demonstrated by the %RSDs being within 0.5-12% for all mycotoxins across all matrices. Results for accuracy and precision are summarised in Table I.
It is important to note that the use of formic acid-containing extraction solution was necessary to obtain adequate recovery for all three fumonisin B compounds (except for tomato puree), but it resulted in low recovery (24-36%) of citrinin in solid food samples.
A simple sample preparation and a fast analysis on a Raptor Biphenyl column under acidic conditions provide a unique solution for mycotoxin analysis that allows simultaneous determination of Alternaria toxins and ergot alkaloid epimers along with other major mycotoxins. Excellent chromatographic results were obtained – including complete separation of all six ergot alkaloids and their epimers – and the method was demonstrated to be rugged, accurate and precise for quantitative determination of mycotoxins in a wide variety of food products.
Dr Shun-Hsin Liang & Dr Jamie York are with Restek