Quantitation of Free and Total N-Acetylcysteine Amide and Its Metabolite N-Acetylcysteine in Human Plasma Using Derivatization and Electrospray LC-MS/MS

摘要

The present invention includes a method of detecting free and total NAC, NACA, or both in a biological sample and/or the effectiveness of a treatment with NAC, NACA, or diNACA comprising: adding 2-chloro-1-methylpyridinium iodide (CMPI) to a biological sample suspected of having NAC or NACA to convert free thiols into stable thioethers; precipitating the protein in the sample; extracting the stable thioethers and separating into a first and a second extract; detecting the thioether derivatives from the first extract with LC-MS/MS; reducing from the second extract free thiols by adding tris(2-carboxyethyl)phosphine (TCEP) followed by converting to stable thioethers with CMPI; detecting the disulfides reduced to free thioether derivatives from the second extract with LC-MS/MS; and calculating from the LC-MS/MS and TCEP of the first and second extracts a free and a total sample NAC or NACA.