Synthesis and spectroscopic characterization of 13C4-labelled 4-cyano-2-oxobutyraldehyde semicarbazone: A metabolite of nitrofurazone

Authors: Singh, Anuradha; Smith, David; Strahan, Gary; Lehotay, Steven

Submitted to: Journal of Labelled Compounds and Radiopharmaceuticals
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/16/2023
Publication Date: 12/3/2023
Citation: Singh, A., Smith, D.J., Strahan, G.D., Lehotay, S.J. 2023. Synthesis and spectroscopic characterization of 13C4-labelled 4-cyano-2-oxobutyraldehyde semicarbazone: A metabolite of nitrofurazone. Journal of Labelled Compounds and Radiopharmaceuticals. 67:18-24. https://doi.org/10.1002/jlcr.4077.
DOI: https://doi.org/10.1002/jlcr.4077

Interpretive Summary: Nitrofurazone was formerly used as an antibiotic feed additive in farm animals including pigs, sheep, goats, cattle, chickens, and turkeys to prevent disease and to promote growth. Due to toxicity concerns, its use in food animals is now prohibited in the U.S. and most U.S. trading partners. Regulatory agencies detect illegal nitrofurazone use by measuring a marker compound called semicarbazide (SEM); however, SEM is produced naturally and has caused numerous false positive test results. Thus, SEM is not a useful marker to detect illegal use of nitrofurazone. A cyano metabolite was proposed as alternative marker of nitrofurazone. Pure chemical standards of the metabolite are not commercially available, so metabolite standards were synthesized, characterized using physical methods (NMR, mass spectrometry), and shared with cooperators at other federal agencies. The work will be useful as it will allow independent groups to validate or refute the usefulness of the cyano metabolite as a marker and will facilitate future research on nitrofurazone fate in farm animals.

Technical Abstract: Nitrofurazone usage in food-producing animals is prohibited in most countries, including the US. Regulatory agencies regularly monitor its use in domestic, export/import animals’ food products by measuring the semicarbazide (SEM) metabolite as a biomarker of nitrofurazone exposure. However, the use of SEM is controversial because it is also produced in food naturally and thus gives false positive tests. A cyano-metabolite, 4-cyano-2-oxobutyraldehyde semicarbazone (COBS), is proposed as an alternate specific marker of nitrofurazone to distinguish nitrofurazone from treated/ or untreated animals. A synthetic method was developed to produce COBS via metallic hydrogenation of nitrofurazone. The product was isolated and characterized by one- and two-dimensional nuclear magnetic spectroscopy (NMR) experiments, fourier-transform infrared spectroscopy (FT-IR), and mass spectrometry. The developed synthetic procedure was further extended to synthesize isotopically labelled 4-[13C]-cyano-2-oxo- [2, 3, 4-13C3]-butyraldehyde semicarbazone. Labelled COBS is useful as an internal standard for its quantification in food-producing animals. Thus, the developed method provides a possibility for its commercial synthesis to procure COBS. This is the first synthesis of the alternate specific-marker metabolite of nitrofurazone for possible usage in regulatory analysis to solve a real-world problem.