Determination of phytochelatins by capillary zone electrophoresis with electrospray tandem mass spectrometry detection (CZE-ES MS/MS)

Abstract

The coupling of capillary zone electrophoresis with electrospray mass spectrometry was optimized for the direct determination of phytochelatins (PCs) in extracts obtained from cells and plants that had been exposed to metal stress. Gluthathione and phytochelatins belonging to the different families (γGlu-Cys)_nGly (n-PC), (γGlu-Cys)_nSer, (γGlu-Cys)_nβAla and (γGlu-Cys)_n were separated in an uncoated capillary at pH 4 using a 5 mM ammonium acetate buffer, and detected by electrospray (ES) MS in the full scan mode (300–1100 u). The use of on-line tandem MS detection in the product ion scan mode of putative protonated molecules of PCs allowed the unambiguous confirmation of the identity of the compounds detected by ES MS. The operational conditions were optimized and the figures of merit were evaluated using n-PC₂, n-PC₃ and n-PC₄ standards purified from a mixture obtained after the reaction of glutathione in the presence of Cd²⁺ and the enzyme PC-synthase. The method was applied to the characterization of bioinduced ligands in cell cultures of soybeans (Glycine max) and in rice (Oryza sativa) roots without the need for a preliminary sample cleanup by size-exclusion and/or reversed phase chromatography.