Here, we provide an extremely simple, rapid, direct, sensitive and specific method to quantify, for the first time in human colon carcinoma cells (HT29) and epidermoid human larynx carcinoma cells (HEp-2), the intracellular glutathione (GSH) using an electrochemical detector coupled to a high performance liquid chromatograph (HPLC-ECD). GSH determination in cancer cells are of valuable interest because GSH has been implicated in the development of resistance to several anticancer drugs. Separation of GSH was performed on a Alltima C18 column using a binary pre-mixed mobile phase of methanol-NaH2PO4 (10 mM, pH 3.0 with phosphoric acid) (2:98, v/v). GSH was detected with an oxidation potential of +500 mV. The limit of detections was 0.30 pMol and good linearity (r = 0.9988) was found. The intra-day and the inter-day recoveries were 97.1–101.3% and 98.2–101.8% respectively, whereas the accuracies were less than 2.56% and 3.02% respectively. The usefulness of the method was established by subjecting HT29 and HEp-2 cells to an oxidant stress generated by menadione, a substance able to decrease GSH level in cells. The experiments show that the HPLC-ECD method developed could represent an useful alternative to the existing procedures since its simplicity and rapidity.

SIMPLE ANALYSIS OF GLUTATHIONE IN HUMAN COLON CARCINOMA CELLS AND EPIDERMOID HUMAN LARYNX CARCINOMA CELLS BY HPLC WITH ELECTROCHEMICAL DETECTION

SPADARO, Angelo;SANTAGATI, Natale Alfredo;RONSISVALLE, Giuseppe
2005

Abstract

Here, we provide an extremely simple, rapid, direct, sensitive and specific method to quantify, for the first time in human colon carcinoma cells (HT29) and epidermoid human larynx carcinoma cells (HEp-2), the intracellular glutathione (GSH) using an electrochemical detector coupled to a high performance liquid chromatograph (HPLC-ECD). GSH determination in cancer cells are of valuable interest because GSH has been implicated in the development of resistance to several anticancer drugs. Separation of GSH was performed on a Alltima C18 column using a binary pre-mixed mobile phase of methanol-NaH2PO4 (10 mM, pH 3.0 with phosphoric acid) (2:98, v/v). GSH was detected with an oxidation potential of +500 mV. The limit of detections was 0.30 pMol and good linearity (r = 0.9988) was found. The intra-day and the inter-day recoveries were 97.1–101.3% and 98.2–101.8% respectively, whereas the accuracies were less than 2.56% and 3.02% respectively. The usefulness of the method was established by subjecting HT29 and HEp-2 cells to an oxidant stress generated by menadione, a substance able to decrease GSH level in cells. The experiments show that the HPLC-ECD method developed could represent an useful alternative to the existing procedures since its simplicity and rapidity.
Column liquid chromatography; Electrochemical detection ; Cell cultures (HT29 and HEp-2); Glutathione
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11769/5742
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