Mycotoxins – Determination of aflatoxins, ochratoxin A, free ochratoxin α, gliotoxin, citrinin, and dihydrocitrinone in urine by LC-MS/MS

Biomonitoring Method – Translation of the German version from 2025

Marion Berger¹ (Method development)
Max Deharde¹ (Method development)
Judith Neuhoff¹ (Method development)
Bernhard Monien² (External verification)
Solveigh Siodlaczek² (External verification)
  Thomas Göen³ (Head of the working group “Analyses in Biological Materials” of the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Deutsche Forschungsgemeinschaft)
  Andrea Hartwig⁴ (Chair of the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Deutsche Forschungsgemeinschaft)
  MAK Commission<sup>5

1</sup> Federal Institute for Occupational Safety and Health (BAuA), Division 4 – Hazardous Substances and Biological Agents, Unit 4.2 – Health Surveillance, Biological Monitoring, Nöldnerstraße 40/42, 10317 Berlin, Germany
² German Federal Institute for Risk Assessment, Unit 54 – Food Safety Department, Max-Dohrn-Straße 8–10, 10589 Berlin, Germany
³ Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute and Outpatient Clinic of Occupational, Social, and Environmental Medicine, Henkestraße 9–11, 91054 Erlangen, Germany
⁴ Institute of Applied Biosciences, Department of Food Chemistry and Toxicology, Karlsruhe Institute of Technology (KIT), Adenauerring 20a, Building 50.41, 76131 Karlsruhe, Germany
⁵ Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Deutsche Forschungsgemeinschaft, Kennedyallee 40, 53175 Bonn, Germany

Abstract

The working group “Analyses in Biological Materials” of the German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) developed and verified the presented biomonitoring method. The aim of this method is the selective and sensitive quantitation of aflatoxins (aflatoxins B1, B2, G1, G2, M1), ochratoxin A (OTA), free ochratoxin α (OTα), gliotoxin (GT), citrinin (CIT) and dihydrocitrinone (DH-CIT) in urine. Sample preparation comprises enrichment and purification of the analytes by solid-phase extraction using OASIS HLB cartridges. Calibration is performed with comparative standards prepared in pooled urine and treated analogously to the samples to be analysed. The aflatoxins, OTA, CIT, and DH-CIT are quantified using isotope-labelled internal standards (ISTDs), whereas OTα and GT are quantified without an ISTD. Determination is carried out by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method provides reliable and accurate analytical results, as shown by the good precision data with standard deviations below 9% for the aflatoxins, OTα, GT and CIT, below 13% for OTA, and below 20% for DH-CIT. Good accuracy data were obtained with mean relative recoveries in the range of 93–107% for the aflatoxins, OTα, GT and CIT, in the range of 83–103% for OTA, and in the range of 81–108% for DH-CIT. The method is both selective and sensitive, and has quantitation limits in the range of 0.013–0.022 μg/l for the aflatoxins and OTA and a quantitation limit of 1.0 μg/l for OTα, 1.5 μg/l for GT, 0.0075 μg/l for CIT, and 0.01 μg/l for DH-CIT.

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