Determination of Alternaria Toxins in Food by SPE and LC-IDMS: Development and In-House Validation of a Candidate Method for Standardisation

Abstract

Alternaria toxins (ATs) are frequently found contaminants in foodstuffs (e.g., alternariol), often reaching high concentrations (e.g., tenuazonic acid). They can spoil a wide variety of food categories (e.g., cereals, vegetables, seeds and drinks) and storage at fridge temperatures does not prevent the growth of Alternaria fungi. Therefore, reliable and validated analytical methods are needed to protect human health and to ensure a transparent and fair trade. This paper describes new technical features that improved a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of ATs in tomato, wheat and sunflower seeds. This analytical method should be simple to implement in different laboratories across the EU and thus be an attractive candidate for standardisation. The major element for improvement was the use of isotopically labelled internal standards, only recently commercially available, thereby reducing the sample handling and improving the accuracy of the results. In addition, the sample extraction and the solid-phase extraction (SPE) enrichment/clean-up were fine-tuned, whereas a more suitable analytical column (XSelect HSS T3) with improved selectivity was also employed. Overall, this method shows adequate precision (repeatability < 5.7% RSD; intermediate precision < 7.0% RSD) and trueness (recoveries ranging from 74% to 112%). The limits of quantification in wheat (the most analytically demanding matrix) vary between 0.19 and 1.40 µg/kg. These figures were deemed satisfactory by the European Committee for Standardization (CEN) and have formed the basis for a subsequent interlaboratory validation study. The corresponding standard was published by CEN in 2021.

Development of a Novel LC-MS/MS Multi-Method for the Determination of Regulated and Emerging Food Contaminants Including Tenuazonic Acid, a Chromatographically Challenging Alternaria Toxin

“…Furthermore, the injection solution consisted of acetonitrile, which caused peak distortion of TEA when the injection volume was higher than 2.0 µL. The separation of Alternaria toxins at pH 8.3 in a cereal matrix gave fit-for-purpose LC-MS/MS analysis ( Figure 4 ), but the high (>50%) ion suppression of AME, described also in earlier methods [ 34 , 35 , 36 , 40 ], was also seen. Therefore, isotope dilution was needed for appropriate quantification.…”

“…Hence, the Alternaria ISTD solution (AME-d3, AOH-d2, TEA- 13 C2) was added to the sample before extraction to enhance the recovery. This was also recommended in previous papers [ 22 , 40 ]. The isotope dilution considerably improved the recovery of TEA, but the 50% loss increased its limit of quantification (LOQ).…”

“…The modification to the standard pesticide method involved the extraction time, which was adjusted to 30 min. Even though QuEChERS has been tested for analysis of Alternaria toxins [ 22 ], the acetonitrile-based extraction was not recommended for the extraction of Alternaria toxins earlier because methanol is preferable [ 40 ]. We also found that the absolute recovery of polar TEA, using QuEChERS extraction, was lower than 50%.…”

Development of Acid Hydrolysis-Based UPLC–MS/MS Method for Determination of Alternaria Toxins and Its Application in the Occurrence Assessment in Solanaceous Vegetables and Their Products

“…Compared with gas chromatography‒mass spectrometry (GC‒MS) and liquid chromatography–diode array detection (LC–DAD), liquid chromatography–tandem mass spectrometry (LC–MS/MS) was the best choice for detecting ATs due to its excellent sensitivity and high efficiency [ 2 , 6 , 8 , 9 , 21 , 22 , 23 ]. However, for sample pretreatment, acetonitrile or formic acid-acetonitrile were the most common extraction solvents following solid-phase extraction (SPE), the QuEChERS (quick, easy, inexpensive, effective, rugged, and safe) method, or direct injection with the isotope internal standard method [ 4 , 19 , 24 , 25 ]. Given the availability of different matrixes, SPE cartridges (HLB) and adsorption materials (GCB and C 18 ) had been optimized and utilized to clean up the samples [ 1 , 5 , 10 , 22 ].…”