Determination of free cyanide (fCN) is required for various industrial, environmental, food, and clinical samples. Enzymatic methods are not widely used in this field despite their selectivity and mild conditions. Therefore, we present here a proof of concept for new spectrophotometric enzymatic assays of fCN. These are based on the hydrolysis of fCN affording the readily detectable NADH. fCN is hydrolyzed either in one step by cyanide dihydratase (CynD) or in two steps by cyanide hydratase (CynH) and formamidase (AmiF). An advantage of the latter route is the higher activity of CynH and AmiF compared to CynD. In both cases, the resulting formate is then transformed by an NAD-dependent formate dehydrogenase (FDH). The NADH thus formed is quantified colorimetrically using a known method based on a reduction of a tetrazolium salt (WST-8) with NADH. The developed assays of fCN are selective except for formic acid interference, proceed under mild conditions, and, moreover, fCN is detoxified during the reactions. The assays proceeded in a microtiter plate format. The limit of detection (LOD) and the limit of quantification (LOQ) were lower for the three-enzyme (CynH-AmiF-FDH) method (7.00 and 21.2 µmol/L, respectively) than for the two-enzyme (CynD-FDH) method (10.7 and 32.4 µmol/L, respectively). In conclusion, the new fCN assays presented in this work are selective, high-throughput, do not require harsh conditions, and use only small amounts of chemicals and enzymes.

Department of Biochemistry Faculty of Sciences Charles University CZ 128 44 Prague Czech Republic

Department of Health Care Disciplines and Population Protection Faculty of Biomedical Engineering Czech Technical University Prague nám Sítná 3105 CZ 272 01 Kladno Czech Republic

Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague CZ 166 28 Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences CZ 142 00 Prague Czech Republic

Anal Bioanal Chem. 2025 Jun;417(14):3229. doi: 10.1007/s00216-025-05853-9. PubMed

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