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Scientifica
Volume 2016, Article ID 6897890, 6 pages
http://dx.doi.org/10.1155/2016/6897890
Research Article

A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells

1Department of Pharmaceutical Chemistry, Sultan Ul Uloom College of Pharmacy, Telangana, Hyderabad 500 034, India
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling, 08100 Bedong, Kedah, Malaysia
3Department of Chemistry, Faculty of Pharmacy, MAHSA University, 59100 Kuala Lumpur, Malaysia
4Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), 42300 Puncak Alam, Selangor, Malaysia
5Faculty of Medicine, AIMST University, Semeling, 08100 Bedong, Kedah, Malaysia

Received 18 December 2015; Revised 6 March 2016; Accepted 7 March 2016

Academic Editor: Qian Wang

Copyright © 2016 Raju N. Appala et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cells. Glutathione is a major intracellular antioxidant having multiple biological effects, best known for its cytoprotective effects against cell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis. Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman’s reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection. The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm. The developed HPLC-UV method was validated with respect to precision, accuracy, robustness, and linearity within a range of 1–20 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively. Furthermore, the method shows the applicability for monitoring the oxidative stress in PC-12 cells.