Multiple methods exist for measuring cyanide in biological fluids. Each method has advantages and disadvantages, and no particular method is used widely and is generally accepted as the gold standard. Thus, improved methods for measuring cyanide in biological fluids are needed, particularly methods that provide for a rapid turn around in the clinical setting.
We have developed a new method to measure cyanide, based on the extremely high affinity of cobinamide for cyanide and the spectral changes that occur when cyanide binds to cobinamide. The assay can be used in both a quantitative mode using a spectrophotometer, and in a qualitative mode by visually observing the color change in cobinamide-impregnated paper. Both methods have a high throughput capacity. The slowest part of the quantitative assay is trapping cyanide in the alkaline cobinamide solution, which requires about 30 min using a Conway microdiffusion cell.
In addition to a high throughput capacity, other advantages of the cobinamide-based method are ease of use, stability of cobinamide, and application across a wide, adjustable dynamic range, depending on the cobinamide concentration used. In contrast to other colorimetric methods such as the NBA/DNB method, which requires two aromatic compounds and an unstable ether, the cobinamide-based method uses non-toxic reagents having less environmental concerns. For the NBA/DNB method, the dynamic range varied based on differing lots of o-dinitrobenzene and on the freshness of the 2-methoxyethanol.
Low cobinamide concentrations were used to validate the new method with respect to a standard method. However, higher concentrations of cobinamide would likely be used in a clinical laboratory, since cyanide concentrations in blood of ~ 40 µM are considered potentially toxic 10
, and a clinical screening modality in the setting of mass casualty should minimize false positives and maximize true positives. While clearly dependent on blood volumes and dilution factors used, cobinamide solutions from 5–100 µM can be used to achieve this degree of sensitivity. The qualitative assay could detect a cyanide concentration > 30 µM in clinical blood samples in 5 min at room temperature, and this might be very useful to rapidly identify cyanide-poisoned patients for early treatment. Moreover, it could be used easily in the field and made quantitative by using a hand-held spectrophotometer or light-emitting diodes.
The major disadvantage of this newly-developed method is the lack of readily available cobinamide. However, it is easy to make from hydroxocobalamin, which is available from multiple suppliers. Dicyanocobinamide is commercially available, but due to cobinamide’s extremely high binding affinity for cyanide, we have found it virtually impossible to remove both cyanide ions quantitatively. Hence the reason it is necessary to make cobinamide from hydroxocobalamin.
While this manuscript was being prepared, Mannel-Croise and Zelder published a paper showing that aquocyanocobinamide could be used to measure cyanide 28
. Because one cyanide molecule is already bound to cobinamide, the dynamic range of the assay is considerably less. Moreover, they applied the method to neat cyanide standards only, and did not test it on biological samples.