Direct determination of glutathione in human blood by micellar electrokinetic chromatography: simultaneous determination of lipoamide and lipoic acid.

A reproducible, rapid procedure for the determination of glutathione in human blood by micellar electrokinetic chromatography has been developed. Whole blood samples were deproteinized with 5% w/v sulfosalicylic acid (final concentration). After centrifugation, the supernatant was directly injected for analysis, without further derivatization. Separations were performed by using an uncoated capillary of 30 cm effective length and 50 microns internal diameter (ID), 50 mM Tris-HCl, 30 mM sodium dodecyl sulfate (SDS), pH 7.00, as running buffer, and 10-20 kV. On-line detection was carried out at 214 nm and a detection limit in the range of femtomoles was achieved. Under the same experimental conditions, we resolved a mixed standard solution containing glutathione in its oxidize and reduced forms, lipoamide and alpha-lipoic acid. The corresponding migration times were reproducible. The present method allows rapid determination of these compounds, which play a critical role in oxidative stress, in cellular defense against injurious agents and whose levels are related to the toxicology and metabolism of several toxins and drugs, such as antineoplastic agents.