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

Appala RN, Chigurupati S, Appala RV, Krishnan Selvarajan K, Islam Mohammad J - Scientifica (Cairo) (2016)

Bottom Line: 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.Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, Sultan Ul Uloom College of Pharmacy, Telangana, Hyderabad 500 034, India.

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.

No MeSH data available.


Related in: MedlinePlus

Representative chromatogram from 6 replicates is shown. 2-Nitro-5-mercapto-benzoic (NMB) acid, glutathione dimer (GSH Dimer), and Ellman's reagent (DTNB) in GSH raw material.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4834400&req=5

fig2: Representative chromatogram from 6 replicates is shown. 2-Nitro-5-mercapto-benzoic (NMB) acid, glutathione dimer (GSH Dimer), and Ellman's reagent (DTNB) in GSH raw material.

Mentions: A segmented gradient program (Table 1) was used to achieve separation with the retention times (RT) of NMB, GSH Dimer, and DTNB at 7.65, 11.23, and 14.83, respectively (Figure 2). When the developed method was tested on samples extracted from PC-12 cells, no endogenous interfering peaks were observed in the individual blank sample at the RT of GSH biosample, making the developed method of high ruggedness.


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

Appala RN, Chigurupati S, Appala RV, Krishnan Selvarajan K, Islam Mohammad J - Scientifica (Cairo) (2016)

Representative chromatogram from 6 replicates is shown. 2-Nitro-5-mercapto-benzoic (NMB) acid, glutathione dimer (GSH Dimer), and Ellman's reagent (DTNB) in GSH raw material.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4834400&req=5

fig2: Representative chromatogram from 6 replicates is shown. 2-Nitro-5-mercapto-benzoic (NMB) acid, glutathione dimer (GSH Dimer), and Ellman's reagent (DTNB) in GSH raw material.
Mentions: A segmented gradient program (Table 1) was used to achieve separation with the retention times (RT) of NMB, GSH Dimer, and DTNB at 7.65, 11.23, and 14.83, respectively (Figure 2). When the developed method was tested on samples extracted from PC-12 cells, no endogenous interfering peaks were observed in the individual blank sample at the RT of GSH biosample, making the developed method of high ruggedness.

Bottom Line: 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.Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, Sultan Ul Uloom College of Pharmacy, Telangana, Hyderabad 500 034, India.

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.

No MeSH data available.


Related in: MedlinePlus