Development of a HPLC-UV method for the simultaneous determination of intracellular glutathione species in human cells

In the present work, an HPLC-UV method was set-up to allow the simultaneous quantification of the reduced-GSH, oxidised-GSSG and nitroso-GSNO glutathione species. Chromatographic separation was achieved on YMC ODS-A C18 column (150 × 4.6 mm, 5 μm), coupled to a Guard-c precolumn (YMC-Pack, 10 × 1-4,0 mm). The eluted compounds were detected at 215 nm by UV-detector, by keeping the column oven at room temperature while the auto-sampler temperature was maintained at 4°C. A fractional factorial design has been applied for the optimization of the mobile phase resulting in baseline separated peaks within 6 minutes. In-house validation was evaluated by linearity, limits of detection (LODs), limits of quantification (LOQs), reproducibility, repeatability and recovery. The detection and quantification limits obtained for standard solutions were below 0.2 μM and 0.6 μM, respectively (RSD values below 2%). The developed method was applied to the measurement of GSH, GSSG and GSNO in human pulmonary cells (A549) exposed to limonene, limonene oxide solubilized into the culture medium and to NO2 as gas phase. Results show an increase in GSH levels, without significant changes in GSSG, when cells were exposed to limonene oxide, while cells exposed to NO2 resulted in a significant increase of GSNO amount. Detection limits were of 1 μM for the glutathione species measured in A549 cells, with RSD values below 2.5%. In conclusion, the present HPLC-UV method can be readily used to measure in a rapid, simultaneous and accurate way the status of GSH, GSSG and GSNO in human cells, their simultaneous quantification helping to better predict the potential impact of chemicals on human health.JRC.I.1-Chemical Assessment and Testin