Determination of Hydroxocobalamin and Cyanocobalamin by Derivative Spectrophotometry in Cyanide Poisoning

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Abstract
Hydroxocobalamin (OHCo) and cyanocobalamin (CNCo) are determined directly in biological media, without extraction, by using first derivative spectrophotometry. We diluted 200 µL of plasma, urine, or standards with 1.8 mL of pH 6 buffer (boric acid, potassium dihydrogen orthophosphate, and potassium hydroxide). The first derivative spectra of the dilutions were plotted between 320 and 400 nm. At the exact zero-crossing point for hydroxocobalamin, the derivative values of cyanocobalamin concentration were determined. The same procedure was followed for hydroxocobalamin at the zero-crossing point for cyanocobalamin. The derivative values of the concentration curves are linear in the range 5–100µM. The minimum detection limit is approximately 5µM for hydroxocobalamin and cyanocobalamin. We have noticed no interference of cyanocobalamin on the determination of hydroxocobalamin or vice versa, although the spectra strongly overlap. The method is fast and simple to use, thus making it easy to assess the in vivo transformation of hydroxocobalamin into cyanocobalamin after the administration of high doses of hydroxocobalamin in cyanide poisoning.

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