Development of a HPLC-UV Method for the Simultaneous Determination of Intracellular Glutathione Species in Human Cells
- *Corresponding Author:
- Barrero-Moreno J
European Commission, Joint Research Centre
Institute for Health and Consumer Protection
Chemical Assessment and Testing Unit
via E. Fermi 2749, I-21027
Ispra (VA), Italy
E-mail: [email protected]
Received date: July 02, 2015; Accepted date: July 20, 2015; Published date: July 27, 2015
Citation: Lipsa D, Cacho C, Leva P, Barrero-Moreno J, Aguar P (2015) Development of a HPLC-UV Method for the Simultaneous Determination of Intracellular Glutathione Species in Human Cells. J Anal Bioanal Tech 6:259. doi: 10.4172/2155-9872.1000259
Copyright: © 2015 Lipsa D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.