Determination of Reduced Glutathione in Human Plasma by RP-HPLC

Aim To establish an HPLC method for the determination of reduced glutathione(GSH) in human plasma.Methods Blood samples were injected into heparinized tubes pre-treated with fixed quantity dithiothreitol(DTT).10% perchloric acid was added to precipitate the proteins.O-phthalaldehyde(OPA) was used as derivative agent of GSH.The derivation was carried out in boric acid buffer.20μl of the supernatant liquid was injected into the HPLC system.The samples were separated on a C18 chromatogram column using a mobile phase consisting of 0.02mol·L-1 KH2PO4 buffer and Acetonitrile(V∶V=94∶6).The flow rate was 1ml/min.The excitation and emission wavelength were set at 340nm and 420nm respectively.Results The standard curve was linear over the range of 0.5 to 200mg·L-1(r=0.996 8,n=7).The intra-day and inter-day RSDs were below 9%.The recoveries of the control samples of low,medium and high concentrations were above 95%.Conclusions The method is accurate and suitable for the research of tforage croppharmacokinetic and the bioequivalence in humans.