Quantitation of cyanide detoxification product ß-cyanoalanine by LC-MS/MS in plant tissue and mitochondrial preparation

Abstract

β-cyanoalanine is a metabolite in the detoxification of cyanide created as a co-product in ethylene biosynthesis pathway. This reaction is catalyzed by β-cyanoalanine synthase (CAS) in the mitochondrion in the presence of cysteine as the other reactant. However, quantitative analysis of β-cyanoalanine by high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) has not yet been demonstrated in plant mitochondria and tissues.

In this study, pear (Pyrus communis) mesocarp and mungbean (Vigna radiata) seedlings as well as their isolated tight-coupling mitochondria were treated with exogenous potassium cyanide (KCN), L/D-cysteine and CAS inhibitor aminooxyacetic acid (AOA). The mitochondrial intactness was supported on the basis of ADP coupling respiration assay. A cation exchange chromatography was utilized for purification of the target compound and couples of parameters were optimized. The standard addition method (SAM) was applied to compensate the matrix effect which usually hampers the quantification of LC-MS/MS. The quantitative results for β-cyanoalanine showed that exogenously applied KCN and L-cysteine yielded a relatively large amount of β-cyanoalanine compared to the control group. AOA inhibited CAS at both tissue and mitochondrial levels; however in the mitochondrial preparation the CAS was totally inhibited whereas in tissue the reduction of the yield of β-cyanoalanine was less than 45%. In mitochondria, D-cysteine in place of the L- form resulted in a sharp drop of β-cyanoalanine (by over 95%). At the tissue level, in contrast, the decrease was not significant. The yield of β-cyanoalanine varied among species. The variation caused by AOA and D-cysteine among mitochondria and tissue level were consistent between pear and mungbean. The whole methodology, composed of the plant materials treatment, extraction and purification process and HPLC-MS/MS analysis can be applied in the quantitative analysis of plant β-cyanoalanine.