Ethoxyquin – Determination of 2,2,4-trimethyl-6(2H)-quinolinone in urine by UPLC-MS/MS

Biomonitoring Method – Translation of the German version from 2026

Gerhard Scherer¹ (Method development)
Nikola Pluym¹ (Method development)
Max Scherer¹ (Method development)
Nadine Rögner¹ (Method development)
Markus Stoeckelhuber¹ (Method development)
Thomas Jäger² (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> ABF Analytisch-biologisches Forschungslabor GmbH, Semmelweisstraße 5, 82152 Planegg, Germany
² BASF SE, Corporate Health Management, Carl-Bosch-Straße 38, 67056 Ludwigshafen, 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 this biomonitoring method for the determination of the most important urinary metabolite of ethoxyquin – 2,2,4‑trimethyl‑6(2H)-quinolinone (EQI). Ethoxyquin is a quinoline-based synthetic antioxidant. Its use as active substance in pesticides was prohibited in 2011. Nevertheless, it continues to be used as a feed additive and is mainly introduced into the environment by feeding treated fishmeal to fish in aquaculture. A human metabolism study showed that ethoxyquin is first metabolised to 1,2‑dihydro-2,2,4‑trimethyl-6‑quinolinol (hydroxyquin) and is then further oxidised to the more stable EQI. In the biomonitoring method presented here, the glucuronide of hydroxyquin in the urine sample is enzymatically hydrolysed using β‑glucuronidase from E. coli. Thereafter, hydroxyquin oxidises spontaneously to EQI. The hydrolysate is subject to salt-assisted liquid-liquid extraction (SALLE) with ethyl acetate. Analysis is performed by UPLC‑MS/MS after positive electrospray ionisation (ESI+). Calibration is performed using standards prepared in pooled urine and processed analogously to the samples. EQI‑D₁₀ is applied as an internal standard. The method provides reliable and accurate analytical results, as shown by the good precision data with standard deviations no greater than 6%. Good accuracy data were obtained with mean relative recoveries in the range of 103–110%. The method is both selective and sensitive, whereby a quantitation limit of 0.03 μg EQI/l was achieved.

---