Development of analytical methods for the determination of tenuazonic acid analogues in food commodities

• Analogues of the Alternaria mycotoxin tenuazonic acid were synthesized.
• Two LC–MS/MS methods were developed for their analysis in food commodities.
• The method based on derivatization with 2,4-dinitrophenylhydrazine showed high sensitivity.
• [13C6,15N]-tenuazonic acid was used as internal standard for quantitation.
• Analogues derived from leucine and valine occurred i.a. in sorghum based infant food.

Analogues of the Alternaria mycotoxin tenuazonic acid (TA, biosynthesized by the fungus from the amino acid isoleucine) derived from valine (ValTA), leucine (LeuTA), alanine (AlaTA) and phenylalanine (PheTA) were synthesized and characterized by mass spectrometry (MS) and 1H nuclear magnetic resonance (NMR) spectroscopy. Concentrations of stock solutions were determined by quantitative 1H NMR (qHNMR). Two analytical methods based on high performance liquid chromatography (HPLC) and MS detection were developed, one with derivatization with 2,4-dinitrophenylhydrazine (DNPH) and one without derivatization. Limits of detection (LODs) were between 1–3 μg/kg (with derivatization) and 50–80 μg/kg (without derivatization). Respective limits of quantitation (LOQs) were about three times higher. Beside TA, the analogues LeuTA (about 4% of TA content) and ValTA (about 10% of TA content) were found in highly contaminated sorghum infant cereals and sorghum grains. Other analogues were not detected. Quantification of LeuTA and ValTA was performed using [13C6,15N]-TA as internal standard and matrix matched calibration. Recovery was between 95 ± 11% and 102 ± 10% for both compounds. Precision (relative standard deviation of triplicate sorghum cereal analyses three times during 3 weeks) was 7% for TA (912 ± 60 μg/kg), 17% for LeuTA (43 ± 8 μg/kg) and 19% for ValTA (118 ± 22 μg/kg). These results indicate that several TA-like compounds, which are not yet characterized in aspects of their toxic properties, were detected in sorghum based infant food highly contaminated with TA, already.