Development and Validation of an LC-MS-MS Method for the Quantification of Cyanate in Rat Plasma and Its Application to Toxicokinetic Bioanalysis

Abstract

Cyanate has been recognized as a uremic toxin that can adversely affect the clinical status of patients with chronic kidney disease. Besides, its toxicity has been under investigation in mammalian toxicology. If such studies are supplemented with toxicokinetic sampling and bioanalysis, additional information can be acquired about the systemic exposure. In order to serve this need, a liquid chromatography with tandem mass spectrometry (LC-MS-MS) method was elaborated and validated for the quantification of cyanate in rat plasma using its isotope-labeled analog for internal standard. Cyanate was converted to a product compatible with reverse-phase LC-MS-MS via a two-step derivatization reaction with the reagent-anthranilic acid. It was observed that this reagent solution contains the reaction products even if prepared freshly in ultrapure water. The phenomenon was interpreted as the presence of urea and its reactivity with anthranilic acid. Contrary to previous research results where fresh anthranilic acid solution was recommended to use, we have found that the aging of the reagent solution is a crucial factor to eliminate the interference. Thereafter, the optimal pH was selected for the plasma sample and processing conditions. Bioanalytical validation and incurred sample reanalysis confirmed the reliability of the method when the intermediate reaction product was used for detection. Only one freeze-thaw cycle stability could be proven, which highlighted the need to collect two sample aliquots whenever possible. Real samples were analyzed in a toxicity study to evaluate systemic exposure of potassium cyanate at three dose levels. Further on, this method might be adapted to provide additional information about the pathophysiological concentration of cyanate in patients with chronic kidney disease for therapeutic support.