CHROMATOGRAPHY
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.
Table 1. Overall range of results across all mycotoxins by matrix (excludes citrinin)
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. Te 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
Fig. 1. Comprehensive mycotoxin analysis under acidic conditions using a Raptor Biphenyl column. Column: Raptor Biphenyl; Dimensions: 100mm x 2.1mm ID; Particle Size: 2.7 μm; Guard column: Raptor Biphenyl EXP guard column cartridge 5mm, 2.1mm ID, 2.7 µm; Temp.: 60°C. Diluent: water (50%) + methanol (50%); Conc.: 0.05–50ng/mL. Inj. Vol.: 5 μL. Mobile phase: A. water + 0.05% formic acid, B. methanol + 0.05% formic acid. Gradient (%B): 0.00 min (25%); 5.00 min (50%); 9.00 min (100%); 9.01 min (25%); 11.0 (25%). Flow: 0.4 mL/min; Detector: MS/MS; Interface: ESI+. Instrument: UHPLC. Complete sample preparation steps and ion transitions are available at
www.restek.com/LC-FS0550
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.
www.restek.com
www.scientistlive.com 27
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