Determination of glutathione isopropyl ester in rat, dog and human blood by high-performance liquid chromatography with fluorescence detection.

A HPLC method is developed for the determination of glutathione isopropyl ester, a drug for the treatment of cerebral vascular disease, in rat, dog and human blood. The blood is deproteinized with sulphosalicylic acid and the clear supernatant treated with a thiol-specific fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F) in borate buffer pH 7.5 at 30 degrees C. The derivatization of glutathione isopropyl ester with SBD-F is markedly enhanced by the addition of dimethyl sulphoxide, and is complete in 30 min. The fluorescent derivatives of glutathione isopropyl ester and the internal standard, glutathione ethyl ester are separated from those of endogenous thiols such as cysteine and glutathione on a reversed-phase column. The method is simple and selective with a detection limit of 0.05 micrograms ml-1. Blood concentrations of glutathione isopropyl ester in rats, dogs and humans after intravenous administration are determined using the method.