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Development of a high-performance liquid chromatography method for the simultaneous quantitation of glutathione and related thiols

Journal Article


Abstract


  • The development of drugs with the ability to increase the level of the antioxidant glutathione and related metabolites has become an important research area for many age-related diseases. Here we describe a high-performance liquid chromatography (HPLC) method that uses the thiol-specific, fluorogenic reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) for the simultaneous determination of total glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), and homocysteine (Hcys) in cell culture medium. ABD-F-labeled thiols were separated using an isocratic mobile phase consisting of 14% methanol and 86% 0.1 M acetate buffer at pH 4.0. The method was validated for linearity, accuracy, and intra- and interday precision, and the lower and upper limits of quantitation (LLOQ and ULOQ, respectively) were determined using a Dionex RF-2000 detector set to medium sensitivity. In addition, the suitability of N-acetylcysteine (NAC) as an internal standard was evaluated by external and internal standard calibration methods. Although both calibration methods showed acceptable linearity (correlation coefficients > 0.99) and intra- and interday precision (relative standard deviations = 10.2 and 6.6%, respectively), the external standard calibration method performed better in terms of accuracy (recovery = 93.7–125%) and also had lower LLOQ values for all analytes (Cys = 6.3 μM, CysGly = 0.8 μM, Hcys = 0.8 μM, and GSH = 1.6 μM).

Authors


  •   Steele, Megan L. (external author)
  •   Ooi, Lezanne
  •   Münch, Gerald (external author)

Publication Date


  • 2012

Citation


  • Steele, M. L., Ooi, L. & Münch, G. (2012). Development of a high-performance liquid chromatography method for the simultaneous quantitation of glutathione and related thiols. Analytical Biochemistry, 429 (1), 45-52.

Scopus Eid


  • 2-s2.0-84864756075

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/4652

Has Global Citation Frequency


Number Of Pages


  • 7

Start Page


  • 45

End Page


  • 52

Volume


  • 429

Issue


  • 1

Place Of Publication


  • United States

Abstract


  • The development of drugs with the ability to increase the level of the antioxidant glutathione and related metabolites has become an important research area for many age-related diseases. Here we describe a high-performance liquid chromatography (HPLC) method that uses the thiol-specific, fluorogenic reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) for the simultaneous determination of total glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), and homocysteine (Hcys) in cell culture medium. ABD-F-labeled thiols were separated using an isocratic mobile phase consisting of 14% methanol and 86% 0.1 M acetate buffer at pH 4.0. The method was validated for linearity, accuracy, and intra- and interday precision, and the lower and upper limits of quantitation (LLOQ and ULOQ, respectively) were determined using a Dionex RF-2000 detector set to medium sensitivity. In addition, the suitability of N-acetylcysteine (NAC) as an internal standard was evaluated by external and internal standard calibration methods. Although both calibration methods showed acceptable linearity (correlation coefficients > 0.99) and intra- and interday precision (relative standard deviations = 10.2 and 6.6%, respectively), the external standard calibration method performed better in terms of accuracy (recovery = 93.7–125%) and also had lower LLOQ values for all analytes (Cys = 6.3 μM, CysGly = 0.8 μM, Hcys = 0.8 μM, and GSH = 1.6 μM).

Authors


  •   Steele, Megan L. (external author)
  •   Ooi, Lezanne
  •   Münch, Gerald (external author)

Publication Date


  • 2012

Citation


  • Steele, M. L., Ooi, L. & Münch, G. (2012). Development of a high-performance liquid chromatography method for the simultaneous quantitation of glutathione and related thiols. Analytical Biochemistry, 429 (1), 45-52.

Scopus Eid


  • 2-s2.0-84864756075

Ro Metadata Url


  • http://ro.uow.edu.au/scipapers/4652

Has Global Citation Frequency


Number Of Pages


  • 7

Start Page


  • 45

End Page


  • 52

Volume


  • 429

Issue


  • 1

Place Of Publication


  • United States