A High-performance Liquid Chromatographic Assay for Reduced and Oxidised Glutathione in Embryonic, Neonatal, and Adult Tissues Using a Porous Graphite Electrochemical Detector

A high-performance liquid chromatographic (HPLC) assay employing a porous graphite electrochemical (EC) detector is described for the simultaneous quantification of reduced glutathione (GSH) and oxidised glutathione (GSSG) in embryonic, neonatal, and adult tissues. Samples were prepared by homogenization in 5% trichloracetic acid, centrifugation, filtration of the supernatant, and injection into the HPLC. Separation was achieved isocratically within 16 min on a 15 cm reversed-phase C18 analytical column with a particle size of 5 micron using an inexpensive mobile phase containing 2-propanol and water (2.8:100) with camphorsulfonic acid and phosphoric acid. The respective limits of detection for GSH and GSSG were 1.5 and 3 ng with a 6 microliter sample using a 3:1 signal to noise ratio. In addition to routine samples, the assay was sufficiently sensitive to detect picomole quantities of GSH and GSSG in small samples, such as a single mouse embryo, gestational day 9, weighing approximately 1 mg. The advantages and disadvantages of the method are compared with other assays for GSH and GSSG published in the literature.