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A Protocol for GC-MS Profiling of Chiral Secondary Amino Acids.

Methods Mol Biol

January 2025

Laboratory of Analytical Biochemistry & Metabolomics, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech Republic.

A simple analytical workflow is described for gas chromatographic-mass spectrometric (GC-MS)-based chiral profiling of secondary amino acids (AAs) in biological matrices. The sample preparation is carried out directly in aqueous biological sample extracts and involves in situ heptafluorobutyl chloroformate (HFBCF) derivatization-liquid-liquid microextraction of nonpolar products into hexane phase followed by subsequent formation of the corresponding methylamides from the HFB esters by direct treatment with methylamine reagent solution. The (O, N) HFB-butoxycarbonyl-methylamide AA products (HFBOC-MA) are separated on a Chirasil-L-Val capillary column and quantitatively measured by GC-MS operated in selected ion monitoring (SIM) mode.

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: Interstitial fibrosis/tubular atrophy in kidney transplantation is an unspecific lesion induced by immune and non-immune factors, which determines the progression of chronic kidney disease. Hydroxyproline is an imino acid that is part of the molecule of collagen. The aim of this study was to assess hydroxyproline in urine microvesicles as a marker of fibrosis in the renal transplant patient.

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Deficiency of cofactors for various enzymes can lead to inborn errors of metabolism. These conditions frequently occur as seizures, which lead to permanent brain damage. Newborn screening for biomarkers associated with these disorders can help in early detection and treatment.

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Background: Tuberculosis (TB) is an infectious disease which has long threatened human health, and new molecular diagnostic markers for its diagnosis are urgently needed. The study was designed to analyze the expression of hydroxyproline (HYP) in different specimens of pulmonary TB (PTB) and assess its auxiliary diagnostic value alone or in combination with the TB infection T lymphocyte spot assay (TSPOT.TB).

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