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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

Linearity plot of 2-nitro-5-mercapto-benzoic (NMB) acid and glutathione dimer (GSH Dimer).
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fig4: Linearity plot of 2-nitro-5-mercapto-benzoic (NMB) acid and glutathione dimer (GSH Dimer).

Mentions: Calibration curves were obtained (using least squares method) by plotting the concentration ratio versus the peak area ratio for the analyte (Figure 4). The method showed linearity within the range of 1 to 20 μg/mL with a correlation cost ability greater than 0.998. The LOD was defined as the compound concentration that produces a signal-to-noise (S/N) ratio greater than three and it was found to be 0.05 μg/mL. The limit of quantitation for the assay was evaluated as the concentration ten times to S/N ratio and was found to be 0.1 μg/mL.


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)

Linearity plot of 2-nitro-5-mercapto-benzoic (NMB) acid and glutathione dimer (GSH Dimer).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Linearity plot of 2-nitro-5-mercapto-benzoic (NMB) acid and glutathione dimer (GSH Dimer).
Mentions: Calibration curves were obtained (using least squares method) by plotting the concentration ratio versus the peak area ratio for the analyte (Figure 4). The method showed linearity within the range of 1 to 20 μg/mL with a correlation cost ability greater than 0.998. The LOD was defined as the compound concentration that produces a signal-to-noise (S/N) ratio greater than three and it was found to be 0.05 μg/mL. The limit of quantitation for the assay was evaluated as the concentration ten times to S/N ratio and was found to be 0.1 μg/mL.

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