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Safeguarding our food: Sensitive and selective LC/MS/MS analysis of aflatoxins
By Pete Stone
Agilent LC/MS Application Scientist
Aflatoxins are highly toxic secondary fungal metabolites that can contaminate dry and stored foods such as grains, nuts, and spices. Since they can cause liver damage and cancer in both humans and livestock, reliable and sensitive analytical methods are needed to protect our food supply. While many procedures exist for analysis of the major aflatoxins, triple quadrupole LC/MS/MS has emerged as a highly sensitive and selective option. Scientists at the National Center for Food Safety and Technology (NCFST) recently completed a detailed study of aflatoxin preparation and analysis using an Agilent 6460 Triple Quadrupole LC/MS System – with excellent results.
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Figure 1. Overlaid extracted ion chromatograms from the MRM analysis show the quantifier ion and the two qualifier ions that increase specificity for each aflatoxin. (Enlarge image.)
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Figure 2. These external standard calibration curves show excellent linearity, which helps to ensure reliable quantitation results. (Enlarge image.)
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Figure 3. Experiments compared recoveries and detection limits for the MycoSep and dispersive SPE cleanup methods. (Enlarge image.)
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Very specific analysis
Researchers Yang Chen and Jack Cappozzo at NCFST demonstrated a robust LC/MS/MS method for simultaneous determination of aflatoxins B1, B2, G1, and G2 in four food matrices (corn flour, wheat, peanut, and walnut). They used the following instrumentation for this work:
Chen and Cappozzo investigated the optimum LC/MS/MS conditions for the four aflatoxins. They determined the MS settings for multiple reaction monitoring (MRM) – a triple quadrupole analysis mode that is well known for reliable quantitation of analytes in complex matrices. Their method used a quantifier ion and two qualifier ions, chosen from runs of aflatoxin standards. They could then confirm the presence of aflatoxins when these ions were present in the correct ratios and at the correct chromatographic retention time. As shown in Figure 1, their LC/MS/MS analysis of the structurally similar aflatoxins required less than five minutes. The detailed instrument conditions are available in a free webinar. After they established the instrument conditions, the scientists ran calibration standards of the four aflatoxins, which produced the linearity shown in Figure 2.
Inexpensive cleanup method with good recoveries
Chen and Cappozzo also studied two rapid sample cleanup methods in conjunction with the LC/MS/MS analysis – the widely used MycoSep cleanup and a less expensive dispersive solid phase extraction (SPE) cleanup (Figure 3).
In this comparison, the investigators found that the inexpensive dispersive SPE cleanup produced comparable results to the established MycoSep technique, with no significant loss in analytical performance. Aflatoxins B1, B2, G1, and G2 showed very low limits of detection (LODs), ranging from 30 to 150 pg/g for all four food matrices, across both cleanup methods. These LODs are all less than 1 pg on-column, attesting to the high sensitivity of the LC/MS/MS system.
Studies from seven sample batches showed aflatoxin recoveries typically between 95 and 110% for both sample cleanup methods. With dispersive SPE cleanup, the walnut samples showed lower recovery (83 to 90%), probably due to ion suppression from matrix interference. By using isotopically labeled internal standards, the investigators improved this recovery to the 95 to 99% range.
Low LODs and few interferences
This study showed that the sample preparation and LC/MS/MS method for aflatoxins B1, B2, G1, and G2 provided low detection limits, high recoveries, and a very selective analysis. The triple quadrupole method is ideal as a stand-alone analysis, or as a confirmation for a previous screen, such as LC with fluorescence detection. If your interest is food safety, learn more about this comprehensive study at NCFST by viewing the free webinar.
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