Determination of Alternaria Toxins in Sunflower Oil by Liquid Chromatography Isotope Dilution Tandem Mass Spectrometry

Abstract

Alternaria toxins have gained attention as a potential health risk and can be classified as emerging mycotoxins. As a result, they are candidates to be regulated by the European Commission. This paper describes a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for analyzing five Alternaria toxins in sunflower oil, which is a rather different type of sample to those matrices investigated in earlier published papers. An optimal sample preparation condition was achieved when samples were dissolved in n-hexane and extracted with methanol/water mixture, followed by sample pre-concentration with solvent evaporation. This study is the first focusing only on this lipophilic matrix and in using all corresponding isotopically labeled internal standards (ISTD) to compensate the matrix effect that strongly influences the LC-MS/MS analysis of toxins. Target compounds were separated on Zorbax Extend C-18 column enabling the analysis at alkaline pH of 8.8 that was necessary to obtain appropriate peak shape of tenuazonic acid and to separate the analytes at baseline. The method was validated according to the EU 2002/657/EC Decision and all the analytical performance characteristics met the requirements. The recovery was between 74% and 122% in fortified sunflower oil samples and the precision varied from 9% to 22%. The method was successfully demonstrated for sunflower seed quality check (QC) samples. Finally, 16 different sunflower oil samples were measured; and tenuazonic acid and tentoxin toxins were detected at levels close to LOQ concentrations.

Levels of Alternaria Toxins in Selected Food Commodities Including Green Coffee

“…AOH, AME, and TeA exhibited a predominant deprotonated molecular ion [M − H] − whereas TEN and ALT were seen either as [M + H] + or [M − H] − species. To avoid the use of polarity switching that may hamper method performances when using older LC-MS instruments, the negative ionization mode was exclusively selected as done previously [20]. Collision-induced dissociation (CID) mass spectra of each precursor were then recorded at various collision energies before selecting the optimal fragment ions, MS/MS transition reactions, and related instrument parameters.…”

“…MRM transition reactions of each internal standard (IS) were the ones corresponding to the related native toxin taking into account the degree of isotope labeling. Adequate LC separation of the five Alternaria toxins was attained with a classical Waters Acquity BEH C18 column employing a 11-min LC gradient in alkaline conditions as done in other studies [5,8,20,21,23]. Mobile phases were ammonium acetate (5 mM) in water pH 8 and methanol.…”

“…Therefore, there is a need for reliable analytical methodologies applicable to a broad range of food commodities. In that context, the European Commission Joint Research Centre (JRC, Geel, Belgium), which was the EU Reference Laboratory for Mycotoxins at that time, organized a Multi Laboratory Test (MLT) in 2018 to support a LC-MS/MS method standardization by the European Committee for Standardization [19,20]. Although limited to the analysis of tomato purees, wheat flour, and sunflower seeds, its analyte scope (AOH, AME, TeA, TEN, and ALT) and quantification approach (isotope dilution) made this confirmatory method a good candidate for deployment in high routine environments, facing today a broad range of items to monitor, an increasing pressure to shorten turn-around time, and cost constraints [21].…”

“…Although limited to the analysis of tomato purees, wheat flour, and sunflower seeds, its analyte scope (AOH, AME, TeA, TEN, and ALT) and quantification approach (isotope dilution) made this confirmatory method a good candidate for deployment in high routine environments, facing today a broad range of items to monitor, an increasing pressure to shorten turn-around time, and cost constraints [21]. Already published methodologies did not match with these expectations due to (a) limited number of food items considered [20], (b) limited analyte scope missing important Alternaria toxin(s) [11], (c) cumbersome sample extraction [14], or (d) quantification approach hardly workable for "multi-matrix" analytical method [8,15,[22][23][24].…”

Food and Drug Analysis

“…Tölgyesi et al [12] described a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for analyzing five Alternaria toxins in sunflower oil. An optimal sample preparation condition was achieved when samples were dissolved in n-hexane and extracted with methanol/water mixture, followed by sample pre-concentration with solvent evaporation.…”