Study of disulfide reduction and alkyl chloroformate derivatization of plasma sulfur amino acids using gas chromatography-mass spectrometry |
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Authors: | Svagera Zdeněk Hanzlíková Dagmar Simek Petr Hušek Petr |
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Institution: | (1) Institute of Clinical Biochemistry, Faculty Hospital Ostrava, 17, Listopadu 1790, 708 52 Ostrava, Czech Republic;(2) Department of Biomedical Sciences, Faculty of Medicine, University of Ostrava, Dvoř?kova 7, 701 03 Ostrava 1, Czech Republic;(3) Biology Centre, Laboratory of Analytical Biochemistry, Branišovsk? 31, 370 05 Česk? Budějovice, Czech Republic |
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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. |
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