A capillary zone electrophoresis for determination of thiolic peptides in biological samples.
A new method to improve the analyses of thiolic peptides (cysteine, gammaGlu-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 microm 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 microM respectively.
Pérez-Rama
Mónica
M
Departamento de Biología Celular y Molecular, Laboratorio de Microbiología, Facultad de Ciencias, Universidade da Coruña, La Coruña, Spain. mperez@udc.es
Abalde
Julio
J
Herrero
Concepción
C
Suárez
Cristina
C
Torres
Enrique
E
D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D056890:Eukaryota / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000032:analysis*
D010455:Peptides; D012997:Solvents; D013438:Sulfhydryl Compounds