No abstract

Four disulfide-reducing agents, dithiothreitol (DTT), 2,3-dimercaptopropanesulfonate (DMPS), and the newly tested 2-mercaptoethanesulfonate (MESNA) and Tris(hydroxypropyl)phosphine (THP), were investigated in detail for release of sulfur amino acids in human plasma. After protein precipitation with trichloroacetic acid (TCA), the plasma supernatant was treated with methyl, ethyl, or propyl chloroformate via the well-proven derivatization-extraction technique and the products were subjected to gas chromatographic-mass spectrometric (GC-MS) analysis. All the tested agents proved to be rapid and effective reducing agents for the assay of plasma thiols. When compared with DTT, the novel reducing agents DMPS, MESNA, and THP provided much cleaner extracts and improved analytical performance. Quantification of homocysteine, cysteine, and methionine was performed using their deuterated analogues, whereas other analytes were quantified by means of 4-chlorophenylalanine. Precise and reliable assay of all examined analytes was achieved, irrespective of the chloroformate reagent used. Average relative standard deviations at each analyte level were ≤6%, quantification limits were 0.1-0.2 μmol L(-1), recoveries were 94-121%, and linearity was over three orders of magnitude (r(2) equal to 0.997-0.998). Validation performed with the THP agent and propyl chloroformate derivatization demonstrated the robustness and reliability of this simple sample-preparation methodology.

No abstract

A simple analytical workflow is described for gas chromatographic-mass spectrometry (GC-MS)-based metabolomic profiling of protic metabolites, particularly amino-carboxylic species in biological matrices. The sample preparation is carried out directly in aqueous samples and uses simultaneous in situ heptafluorobutyl chloroformate (HFBCF) derivatization and dispersive liquid-liquid microextraction (DLLME), followed by GC-MS analysis in single-ion monitoring (SIM) mode. The protocol involves ten simple pipetting steps and provides quantitative analysis of 132 metabolites by using two internal standards. A comment on each analytical step and explaining notes are provided with particular attention to the GC-MS analysis of 112 physiological metabolites in human urine.