Optimization of the principal parameters for the ultrarapid electrophoretic separation of reduced and oxidized glutathione by capillary electrophoresis.
Several factors can influence the analytical efficiency and rapidity of the quantitative determination of erythrocyte glutathione by capillary zone electrophoresis (CZE). We optimized the time, efficiency and resolution of the electrophoretic separation of reduced (GSH) and oxidized (GSSG) glutathione by studying the influence of the most important factors affecting the separation, i.e. the pH and ionic strength of the electrolyte solution, the capillary length and temperature. Best results in the shortest time are obtained at 25 degrees C, using an uncoated 37 cm x 75 microm i.d. capillary and a 300 mmol/l borate buffer pH 7.8. These conditions give a good reproducibility of the corrected peak areas (R.S.D. 1.41 and 1.31%) and of the migration time (R.S.D. 0.22 and 0.26%) for GSH and GSSG, respectively. The high concentration buffer, besides permitting a good resolution of standard GSH and GSSG mix, allows also N-nitrosoglutathione detection. By shortening the capillary length to 27 cm, the separation time of GSH and GSSG can be further decreased to less than 60s. This shortened method, the most rapid described in literature, can detect and quantify GSH in red blood cells despite a loss of sensitivity. To compare the new method here described with the Beutler colorimetric method, the data relative to the GSH content of red blood cells from young normal subjects were analyzed by the Passing and Bablok regression and the Bland-Altman test.
Carru
Ciriaco
C
University of Sassari, 07100 Sassari, Italy. carru@uniss.it
Zinellu
Angelo
A
Sotgia
Salvatore
S
Marongiu
Giovanni
G
Farina
Maria Grazia
MG
Usai
Maria Franca
MF
Pes
Giovanni Mario
GM
Tadolini
Bruna
B
Deiana
Luca
L
D019075:Electrophoresis, Capillary / Q000379:methods*; D005978:Glutathione / Q000097:blood / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D026422:S-Nitrosoglutathione / Q000097:blood
D026422:S-Nitrosoglutathione; D005978:Glutathione