| CPC G01N 33/6848 (2013.01) [G01N 33/6815 (2013.01)] | 6 Claims |
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1. A method of stabilising a biological sample comprising glutathione (GSH) and glutathione disulfide (GSSG), the method comprising the following steps:
a) providing the biological sample comprising GSH and GSSG;
b) contacting GSH and GSSG of the biological sample with a maleimide under conditions which allow alkylation of the sulfhydryl group of GSH with the maleimide to obtain maleimide-alkylated GSH, wherein the maleimide is N-ethyl maleimide (NEM);
c) separating excess maleimide from maleimide-alkylated GSH and GSSG by solvent extraction, wherein the solvent is an inorganic solvent selected from the group consisting of dichloromethane, chloroform, ethyl acetate, hexane, diethyl ether, and mixtures thereof;
d) contacting maleimide-alkylated GSH and GSSG after step c) with a reducing agent selected from the group consisting of tris(2-carboxyethyl) phosphine (TCEP), tributylphosphine, dithiothreitol (DTT), dithioerythritol (DTE) and β-mercaptoethanol, under conditions which allow reduction of GSSG by the reducing agent to obtain further GSH; and
e) contacting maleimide-alkylated GSH and GSH after step d) with a heavy isotopologue of the maleimide under conditions which allow alkylation of the sulfhydryl group of GSH with the heavy isotopologue of the maleimide to obtain a heavy isotopologue of the maleimide-alkylated GSH;
whereby a stabilised biological sample, comprising the maleimide-alkylated GSH and the heavy isotopologue of the maleimide-alkylated GSH, is obtained;
wherein the heavy isotopologue of the maleimide is N-ethyl-D5-maleimide (D5-NEM).
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