Quantification of Intracellular Thiols by HPLC-Fluorescence Detection

Abstract

Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and N-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R2 > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5–153 pmol/1 × 106 cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.

Chemoselective bicyclobutane-based mass spectrometric detection of biological thiols uncovers human and bacterial metabolites

“…47 Prior studies have mainly focused on investigating redox changes related to the common thiol-containing cysteine, glutathione or homocysteine. 35,[48][49][50] Overall, only a few studies have aimed for a broad-scale analysis of biological thiols and were hampered in metabolite coverage due to analytical limitations.…”

A rapid and sensitive colorimetric discrimination and detection of cysteine, homocysteine and glutathione by phloroglucinol-functionalized silver nanoresonators with real applications

“…7,8 Hence, in view of the very important roles of these biothiols, several instrument based methods have been developed for their detection and determination by various researchers. [9][10][11] However, the high cost and difficulty in portability of these instruments are the main bottlenecks in their wide spread application by common people. In the last couple of decades, chemo sensors have come up as the favorite user choice for the real time detection and determination of a variety of analytes including those of bioorigin.…”