A capillary zone electrophoresis for determination of thiolic peptides in biological samples

Abstract

A new method to improve the analyses of thiolic peptides (cysteine, γGlu-Cys, glutathione, phytochelatins and desglycyl-phytochelatins) derivatized with monobromobimane (mBrB) in complex biological samples by CZE is described. The method involves a SPE using Sep-Pak Light C18 Cartridges after derivatization and a later CZE analysis. Elution of mBrB-thiols was achieved with 10 mM HCl + 70% methanol v/v in deionised water. Electrophoretic parameters, such as BGE pH and concentration, different organic additives (methanol and trifluoroethanol), applied voltage and capillary length were studied in order to establish suitable analytical conditions. Optimum separation of the mBrB-thiolic peptides was obtained with 100 mM sodium borate buffer at pH 7.60. The electrophoretic conditions were +15 kV, capillary length of 90 cm from inlet to detector (98 cm total length, 50 μm ID), samples were loaded into the capillary by hydrodynamic injection (50 mbar, 20 s) and detection was performed at 390 nm. The improved method showed good reproducibility, linearity and sensitivity. The LODs and LOQs estimated using a standard of GSH were 1.41 and 4.69 μM respectively.